Citation
Purification of a eukaryotic RNA polymerase II that synthesizes polyadenylic acid

Material Information

Title:
Purification of a eukaryotic RNA polymerase II that synthesizes polyadenylic acid
Creator:
Benson, Robert Henry, 1942-
Publication Date:
Language:
English
Physical Description:
xiii, 112 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Actinomycin ( jstor )
Corn ( jstor )
DNA ( jstor )
Enzymes ( jstor )
Gels ( jstor )
Nucleotides ( jstor )
Precipitation ( jstor )
Purification ( jstor )
RNA ( jstor )
Titration ( jstor )
Dissertations, Academic -- immunology and medical microbiology -- UF ( mesh )
Immunology and Medical Microbiology Thesis Ph.D ( mesh )
Poly A ( mesh )
Polymers ( mesh )
RNA ( mesh )
RNA Polymerase II ( mesh )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1972.
Bibliography:
Includes bibliographical references (leaves 100-104).
Additional Physical Form:
Also available online.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Robert Henry Benson.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
0029717831 ( ALEPH )
24663390 ( OCLC )

Downloads

This item has the following downloads:


Full Text




















PURIFICATION OF A EUKARYOTIC
RNA POLYMERASE II THAT SYNTHESIZES
POLYADENYLIC ACID






By




ROBERT HENRY BENSON













A DISSERTATION PRESENTED TO THE GRADUATE
COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY






UNIVERSITY OF FLORIDA 1972



































This dissertation is dedicated to my parents, MaryAnn L. and Henry E. Benson, for their endless encouragement during my 25 years of education.














ACKNOWLEDGMENTS


I would like to thank Dr. Rusty J. Mans for all the time and effort he has put forth on my behalf, both scientifically and personally. At first I didn't know quite.,how to cealwith his enthusiastic approach to problems, but gradually I learned. I would like to thank .Dr. George E. i(]Ifford for introducing me to microbiology and teaching me that research could be both enjoyable and challenging. it would like to thank Dr. Ira Rosen for introducing me tb genetics and transcription and I would like to thank Dr. Daniel Billen for introducing me to Dr. Rusty J. Mans. As usual, there are all the other people and I would like to thank them too. I would like to thank Muriel Reddish who knows everything that a graduate student ever needs to know about the department. My thanks to Claudia Alverez who shares everyone's laboratory frustrations, especially those of Dr. Rusty J. Mans. My thanks to Sharon Bryant, Bob Brooks, Gary Benson, Carl Smith and Norm Huff for keeping me company in our one-windowed laboratory. I would really like to thank the N. I. H. which has kept me lean but alive during the last five years. To my roommates, Barbara, Joe and Chantal, "thanks, and whose turn is it to do the dishes?" And in ii i










conclusion, I would like to thank John Henry Colson and Bernardine, for keeping me company during all my nights in the laboratory repeating experiments and typing endless drafts of this dissertation.











































jv













TABLE OF CONTENTS


Acknowledgments . . . . . . . . . .

List of Tables . . . . . . . . . . vi

List of Figures . . . . . . . . . . vii

Key to Abbreviations . ... . . . ... . . ix

Abstract . . . . ... . . . . . . xi

Introduction . . . . . . . . . . 1

Literature Review . . . . . . . . . 4

Methods and Materials . . . . . . .. . 21

Results . . . . . . . . . . . . 31

D--..-.ussion . . . . . . . . . . . 86

Conclusion ... . . . . . . . . . . 99

References ... . . .. . . . . . . . 100

Appendix A . . . . . . . . . . . 105,

Appendix B . . . . . . . . . . . 109

Biographical Sketch . . . . .. . . . . 112.














LIST OF TABLES


1. STEPS IN RNA POLYMERASE PURIFICATION ... ....... .32

2. STORAGE AND FREEZE-THAW LABILITY OF RNA
POLYMERASE ......... ..................... 40

3. SALT PRECIPITATION OF RNA POLYMERASE ... ........ 42

4. ASSAY REQUIREMENTS OF RNA POLYMERASE ... ........ 44

5. NEAREST NEIGHBOR FREQUENCY ANALYSIS OF RNA
PRODUCT SYNTHESIZED WITH DENATURED DNA .......... .59

6. AMP AND UMP INCORPORATION BY RNA POLYMERASE . . 65

7. INHIBITION OF POLYADENYLIC ACID SYNTHESIS BY NUCLEOTIDES .................... 67

8. EFFECT OF DELAYED ADDITION OF NTPs ON POLYADENYLIC ACID SYNTHESIS ................. .......... 68

9. EFFECT OF HEATING AND SDS ON RNA POLYMERASE PRODUCTS .......... ......... ......... .... 71
10. NEAREST NEIGHBOR FREQUENCY ANALYSIS OF POLYADENYLIC ACID PRODUCT ...... ............... 72

11. AVERAGE CHAIN LENGTH OF POLYADENYLIC ACID ...... .73 12. INHIBITOR SENSITIVITY OF RNA POLYMERASE .. ...... 76 13. COMPARISON OF CALF THYMUS AND MAIZE DNA AS
TEMPLATES FOR RNA POLYMERASE ..... ............ 82

14. RNA POLYMERASE ACTIVITY WITH SYNTHETIC TEMPLATES. .84









vi














LIST OF FIGURES


1. PURIFICATION PROCEDURE FOR RNA POLYMERASE ... 22 2. DEAE-CELLULOSE GRADIENT ELUTION ASSEMBLE . . 25 3. DEAE-CELLULOSE ELUTION PROFILE. . . . . 34

4. RNA POLYMERASE ACTIVITY IN DEAE-CELLULOSE FRACTIONS . . . . . . . . . 35

5. POLYACRYLAMIDE GEL ELECTROPHORESIS OF NATIVE RNA POLYMERASE . . . . . . . . . 37

6. POLYACRYLAMIDE GEL ELECTROPHORESIS OF DENATURED RNA POLYMERASE . . . . . . . . . 38

7. DNA TITRATIONS WITH MAGNESIUM AS COFACTOR . .. 46 8. DNA TITRATIONS WITH MANGANESE AS COFACTOR. . . 47 9. MAGNESIUM TITRATION WITH NATIVE AND DENATURED DNA. 49 10. MANGANESE TITRATION WITH NATIVE AND DENATURED DNA. 50 11. NTP TITRATION OF. RNA POLYMERASE . . . . 51

12. ATP TITRATION OF RNA POLYMERASE . . . . .. 53

13. LINEWEAVER-BURKE PLOTS OF ATP TITRATIONS . . 54 14. AMMONIUM SULFATE TITRATION OF RNA POLYMERASE . 55 15. RNA POLYMERASE ACTIVITY AS A FUNCTION OF ENZYME
CONCENTRATION . . . . ... . . . . 57

16. RATE OF AMP INCORPORATION AS A FUNCTION OF TIME. 58 17. DNA TITRATIONS WITH MAGNESIUM IN THE ABSENCE OF
NTPs .. . . . . . . . . . . .62

18. DNA TITRATIONS WITH MANGANESE IN THE ABSENCE OF
NTPS . . . . . . . . . . . 63

vii










LIST OF FIGURES (continued)


19. NTP TITRATION WITH DENATURED DNA . . .. 66 20. POLYADENYLIC ACID SYNTHESIS AS A FUNCTION OF
ENZYME CONCENTRATION . . . . . . . 70

21. ALPHA-AMANITIN TITRATION OF RNA POLYMERASE. . 78 22. ACTINOMYCIN D TITRATION OF RNA POLYMERASE . . 79 23. RIBONUCLEASE SENSITIVITY OF DENATURED DNA-DEPENDENT
PRODUCTS . . . . . . . . . 81

24. AMP INCORPORATION AS A FUNCTION OF ATP SPECIFIC
ACTIVITY . . . . . . . . . 106

25. RATE OF AMP INCORPORATION AS A FUNCTION OF ATP
CONCENTRATION . . . . . . . . . 108

26. A MODEL FOR POLYADENYLIC ACID INITIATION OF
TRANSCRIPTION . . . . . . . . . 110

























viii










KEY TO ABBREVIATIONS


A260 absorbancy at 260 rim

ATP adenosine triphosphate

AMP adenosine monophosphate

BSA bovine serum albumin

CAP catabolite gene-activator protein

cpm counts per minute

CTP cytidine triphosphate

d dalton

DMSO dimethyl sulfoxide

dpm disintegrations per minute

DEAt diethylaminoethane

DNA deoxyribonucleic acid

GTP guanosaine triphosphate

g gravity h hour

HrRNA heterogeneous nuclear ribonucleic acid

NTPs GTP, CTP, UTP

POPOP 1,4-bis-,[2- (4-methyl-5-phenyloxazolyl) Ibenzene

PPO 2, 5-diphenyloxazole

poly (A) polyadenylic -acid

poly(dAT) alternating copolymers of deoxyadenylic
and deoxythymidylic acid poly (U) polyuridylic acid

poly(dAdT) homopolymers of deoxyadenylic and
deoxythymidylic acid.

IX










KEY TO ABBREVIATIONS (continued) RNA ribonucleic acid

rRNA ribosomal ribonucleic acid

S sedimentation coefficient

SSC standard saline citrate

SDS sodium dodecyl sulfate

TCA trichloroacetic acid

tris tris(hydroxymethyl)aminomethane

tRNA transfer ribonucleic acid

UTP uridine triphosphate

UMP uridine monophosphate

50% ASP 50% ammonium sulfate precipitate


























x










Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy





PURIFICATION OF A EUKARYOTIC RNA POLYMERASE II
THAT SYNTHESIZES POLYADENYLIC ACID By

Robert Henry Benson

December, 1972



Chairman: Rusty J. Mans, Ph.D. Major Department: Immunology and Medical Microbiology


The in vitro catalytic activities of DNA-dependent RNA polymerase II, isolated and purified from rapidly growing 5-day-old maize seedlings (WF9 x Bear 38, waxy), have been examined. An enzyme purification procedure is described utilizing homogenization, differential centrifugation, salt precipitation and ion exchange chromotography that resulted in 1,000-fold purification with 70 percent recovery in 6 to 10 hours. The enzyme had a specific activity greater than 100 nmoles AMP/mg/20 min. at 30*, and contained one major band and several minor bands on polyacrylamide gel electrophoresis. Incorporation of AMP into acid-insoluble material required DNA, 4 nucleosidetriphosphates: GTP, CTP, UTP, ATP (NTPs),
metl1eihe ~2+ 2+
and metal, either Mn or Mg2. Alkaline hydrolysis of

xi










product, synthesized with [a-32P]ATP, NTPs, denatured DNA and Mn2+, resulted in these nearest neighbor frequencies: CpA, 0.25; ApA, 0.44; GpA, 0.18; UpA, 0.13. The requirements for incorporation of a labeled nucleotide into acidinsoluble material and the nearest neighbor frequency indicate that RNA was synthesized. In the presence of labeled ATP alone, the ApA frequency of alkaline-hydrolyzed product was 0.90, and the AMP/adenosine ration was greater than 100, indicating polyadenylic acid [poly(A)] was synthesized. Since UTP was incorporated in the presence but not in the absence of NTPs, no poly(U) was synthesized, suggesting that poly(A) may be the only homopolymer accumulated. Poly(A) synthesis was 90 percent inhibited by 0.025 mM NTPs, whereas both RNA and poly(A) were synthesized with 1.0 mM (or greater) NTPs, suggesting the mode of poly(A) synthesis differs as a function of the nucleotides present, but all products synthesized, were acidinsoluble after heating (1000) for 5 min. in 1 percent SDS, indicating that product-protein and product-DNA complexes were not responsible for acid-insolubility. RNA and poly(A) synthesis was inhibited by 1 4M e-amanitin, indicating that both products were synthesized by RNA polymerase II. Poly(A) synthesis was supported by poly(dAdT), but not by poly(dAT), suggesting a poly(dT) template was utilized for poly(A) synthesis. Actinomycin D (7 1M) inhibited RNA synthesis 87 percent on denatured calf thymus xii










DNA, but stimulated poly(A) synthesis 28 percent, again suggesting poly(dT) regions of DNA were utilized as a template for poly(A) synthesis. Neither RNA nor poly(A) synthesis was inhibited by cordycepin (0.26 mM) or rifampicin (50 uig/ml), therefore eliminating the presence of maize NTP: exotransferase and bacterial RNA polymerase activity. Since early product (accumulated at a high rate of AMP incorporation) was resistant to pancreatic ribonuclease, whereas, late product (accumulated at a lower rate of AMP accumulation) was sensitive to RNase digestion, poly(A) synthesis apparently -preceded RNA synthesis. A model is presented which requires that poly(A) synthesis precede. RNA synthesis in the same enzyme-template complex during the initiation of transcription in eukaryotes.






















xiii













INTRODUCTION


Macromolecular nucleic acid metabolism depends upon two classes of enzymes: the polymerases which assemble the polymers from the nucleoside or deoxynucleoside triphosphates, and the nucleases which break down the polymers. Among the polymerases there are four types which are known to utilize a nucleic acid as a template to synthesize a new polymer. These are DNA-dependent DNA polymerase (EC 2.7.7.7), DNA-dependent RNA polymerase (EC 2.7.7.6), RNA-dependent RNA polymerase and RNA-dependent DNA polymerase. Of primary importance in phenotypic expression of genetic information are the DNA-dependent RNA polymerases. These enzymes are responsible for the synthesis of messenger RNA,the template for sequencing amino acids, and for the synthesis of ribosomal and transfer RNAs, needed for translation of the messenger RNA into protein. Control of the synthesis of these RNAs seems to be primarily at the level of initiation of transcription.

Transcription may be studied in vivo or in vitro.

The in vitro approach to transcription requires a purified RNA polymerase. Once the enzyme is purified, specific properties can ten be determined, such as the assay


1






2



requirements and product characteristics. Following this initial evaluation, transcriptional control functions, such as template specificity or the presence or absence of initiation and termination factors, can be determined.

The DNA-dependent RNA polymerases all require a DNA template, the four nucleoside triphosphates: ATP, UTP, GTP and CTP, and metal during the synthesis of RNA and concomitant liberation of pyrophosphate. The RNA polymer is synthesized in the 5' to 3' direction. The new nucleotide is linked via a phosphodiester linkage to the 3' hydroxyl of the growing RNA cha.in. As the RNA is synthesized in the 5' to 3' direction, the template DNA is transcribed in the 3' to 5' direction. RNA polymerase activity can be evaluated separately in four operational steps: binding, initiation, elongation and termination. Step one is the binding of the RNA polymerase to the DNA template. -Step two is the initiation of an RNA chain via synthesis of the first phosphodiester linkage. Step three is the elongation of the new RNA chain. Step four is the termination of RNA synthesis. Release of the RNA chain may occur immediately or it may be delayed.

Experimental evaluation of the initial steps,

binding to the DNA template and initiation of RNA synthesis, requires an enzyme~of high specific activity. This high activity is required to experimentally detect






3



the initial synthetic activity of the RNA polymerase and to minimize the influence of any other enzymatic activities present. In addition, experimental manipulation of the enzyme during purification should be kept minimal, to reduce the probability of enzyme degeneration and, therefore, to preserve in vivo transcriptional activity. The experimental approach in this study was to purify the RNA polymerase from maize seedlings, to determine its requirements for product accumulation and to identify the products. RNA polymerase was purified by centrifugation, (NH4)2So4 precipitation and ion exchange column chromatography in that order. From its assay requirements and inhibitor sensitivities it was identified as RNA polymerase type II. In addition to its known catalysis of RNA synthesis, this eukaryotic RNA polymerase catalyes polyadenylic acid synthesis. The polymerase products were characterized by sensitivity to heat, SDS and pancreatic ribonuclease, and by nearest neighbor frequency analysis. The possible involvement of poly(A) synthesis as a mechanism of initiation of transcription is suggested and discussed.













LITERATURE REVIEW



Approaches to Transcription


Guides to Literature

There are several avenues into the literature of

transcription; for current research, the symposia (1,2,3) and reviews (4,5,6) are best, whereas for a more historical perspective, there are collected papers (7) and introductory texts (8,9,10).


Systems Studied

Essentially every level of life from the smallest viruses to the largest eukaryotes has had, or is having, its transcriptional processes studied. The basic enzymology has been done in bacterial systems, principally Escherichia coli and Bacillus subtilis and their phages (11,12). Although RNA polymerase was first detected in a eukaryote by Weiss in 1960 (13), the bacterial viruses and their nucleic acids proved most useful in elucidating transcription in prokaryotic systems (11,14). The major difficulties encountered in studying the eukaryotic systems were the initial low activity of the eukaryotic RNA polymerases and the difficulty in getting the enzyme DNA-dependent (15). These



4







5


problems have been solved and the study of in vitro transcription in eukaryotes is rapidly advancing.

Transcription as a subject may be broken into two

areas of investigation: first, the prokaryotes and their viruses that have served as model systems due to the ease of acquiring enzyme and defined DNA templates; second, the eukaryotes that have complex chromosomes and multiple polymerase.systems producing the intricate specialized tissues of plants and animals. The bacterial systems will be illustrated by-the E. coli and B. subtilis systems and the eukaryotes by the calf thymus and maize systems.





Bacterial Transcription


Significant Concepts

There-are extensive reviews of transcription available (1,2,4,5,6). I would like to draw from these systems to illustrate four concepts which have recently been uncovered in bacterial transcription.


1. RNA'polymerase and associated factors have
the ability to initiate and terminate transcription at specific sequences on the
chromosome.

2. Alteration of the RNA polymerase, either
through viral infection or sporulation,
results in a change in the transcription
specificity of the RNA polymerase.







6


3. RNA polymerase may be the site of hormone
action via specific protein factors such
as CAP (catabolite gene-activator protein) which are influenced by hormone-controlled
cyclic AMP.

4. Alteration of the RNA polymerase transcription
specificity can result in irreversible
changes in cell phenotype, closely
resembling differentiation in eukaryotes.


These four concepts illustrate the potential importance of a better understanding of the RNA polymerases in both prokaryotes and in eukaryotes.


E. Coli RNA Polymerase and Sigma Factor

E. coli RNA polymerase contains four types of subunits: 8' (155-165,000 d), (145-155,000 d), a (85-95,000 d), and (39-41,000 d). A fifth subunit, w (10,000 d), is sometimes found with the RNA polymerase although it is not required for RNA polymerase function (4). Together these subunits are arranged as holenzyme.(a'sa a), which retains
2
the ability to asymmetrically transcribe T4 DNA, or as core enzyme (8'a ), which cannot asymmetrically transcribe T4
2
DNA (11). The presence or absence of sigma (a) determines the initiation specificity of the RNA polymerase on T4 DNA. Another factor, rho (p), although not bound to the RNA polymerase, when present with the enzyme resulted in one type of specific termination of RNA synthesis (15). Two other sites also resulted in chain termination. The RNA polymerase, without rho, also recognized sequences in the DNA transcribed as UAA (ochre) and UAG (amber) and terminated







7


RNA chains (15).

In response to infection by coliphage T4, E. coli

RNA polymerase transcribed early T4 RNA. After production of the gene 55 protein, translated from the early RNA, the synthesis of delayed early RNA was specifically initiated

(16). Travers (17) isolated the protein and called it a T4 sigma-like factor which caused specific asymmetric initiation of T4 DNA at the site of delayed early RNA. This isolation of a factor which directed delayed early RNA synthesis demonstrated for the first time that RNA poly,merase could acquire a new initiation specificity (16,17). One minute after infection by phage T4, the host RNA polymerase no longer synthesized early T4 RNA (18). This change was apparently caused by alteration of the cc subunit of the host RNA polymerase through adenylation with 5AMP (19). Therefore, viral infection initiated changes in the transcription machinery which caused a specific alteration of the initiation sites for RNA synthesis. These changes included alteration of the existing host RNA polymerase and the synthesis of a viral protein to replace the host sigma factor.


E. Coli RNA Polymerase and CAP RNA polymerase control in E. coli illustrates the possible importance of transcription as a mechanism for bor one action. Cyclic AMP, together with CAP, was required for maximum expression of the lactose and other inducible







8


operons (20,21). CAP and cyclic AMP were bound to the lactose repressor protein and catalyzed its dissociation from the operator gene (21). The results with CAP raised the possibility that transcription of many prokaryotic and eukaryotic genes requires the action of an additional positive control element. The requirement for cyclic AMP linked transcriptional control to hormone action (22). B. Subtilis RNA Polymerase and Sporulation

The sporulation of B. subtilis resulted in changes in the template specificity of the RNA polymerase. These changes were associated with a decrease in transcription of vegetative genes and the expression of new sporulation specific genes (23,24). The sporulating cell contained an RNA polymerase with a subunit alteration such that it could not utilize vegetative sigma factor (25). This irreversible change in cell phenotype resembled differentiation, since once sporulation, began, the cell was committed and could not return to vegetative growth except through the sporulation stage.





Eukaryotic Transcription


General Background

While eukaryotic RNA polymerase activity was detected as early as 1960 by Weiss (13), initial progress at










purificatiows.very slow. This was a result of the initi iontatiitby of the RNA polymerase in eukaryotic tissues, of heifi'ulty encountered in freeing the enzyme of conritaminating 'DNA and of the instability of the enzyme during purification (26). These problems were overcome and soluble DNA-dependent RNA polymerases were purified from eukaryotic tissues (27,28,29,30). In 1970, Roeder and Rutter (31) first detected the presence of two types of RNA polymerase, Thype I was a nucleolar RNA polymerase, insensitive to a-amanitin, that synthesized a product which competitively., hybridized with r-RNA but not with Hn-RNA. .Type II was a nuiptoplasmic, a-amanitin sensitive RNA polymerase that synthesized a product that competitively hybridized with Hn-RNA but not with r-RNA (32). This was the first time that specific RNA polymerases were shown to be localized inside a eukaryotic cell and to be responsible for specific classes of eukaryotic RNA. Since then it has been shown that the nucleus, the nucleolus, and the cellular organelles have unique transcriptional systems (15).

Eukaryotes have a complex chromosomal structure

involving nucleic acids, histones, and acidic proteins. The relationship between the histones and differentiation is, uncertain, a though histones are believed to be intimately involved in gene selection (33). There are two major approaches to how this interaction might occur.







10


First, transcription may be activated by removal of histones, somewhat analogous to removal of the repressor on the lactose operon. Second, RNA polymerase and specific initiation factors initiate transcription by opening the genes for transcription, with histone removal occurring as the RNA polymerase precedes (15). Accordingly, either the histones or the RNA polymerases may function to control the specificity of transcription, or a combination of both systems.

Acidic nuclear proteins are of interest to transcription because of their physical properties and location (34). Since the RNA polymerases are themselves acidic nuclear protein complexes, some of the acidic proteins may be subunits of the RNA polymerases or of the other polymerases. The acidic nuclear proteins may contain factors that function along with the histones for gene selection and control of transcription.


Transcription Products

There are at least three classes of RNA produced in all cells: rapidly labeled RNA including HnRNA and mRNA; stable RNA or GC-rich RNA which is rRNA; and soluble or tRNA. These three classes of RNAs constitute the bulk of the transcriptional products in all cells. Following transcription, the RNA is often processed by specific systems which selectively degrade the gene product into the functionally active form. The processing of ribosomal










RNA is the best studied. The ribosomal genes are sequesteed.in the nucleolus and are transcribed as a unit into-a sL.le'40-45 S precursor RNA molecule. This RNA is pTocessed by a series of post-transcriptional cleavage steps to give 18 S and 25 S RNAs found in mature amphibian oocyte ribosomes (35). Heterogeneous RNA is found in the nucleoplasm and has a DNA-like base composition. A large fraction of this rapidly labeled RNA never leaves the nucleus and may be involved with regulation at the level of transcription

(36). Both rRNA and tRNA are stable and constitute the bulk of the RNA contained in a cell at any one time, while most of the rRNA rapidly turns over.


RNA Polymerases

Types

Type I RNA polymerase is localized in the nucleolus and synthesizes GC-rich RNA that competitively hybridizes with ribosomal RNA C32). Its polymerizing activity is resistant to c-amanitin, it is highly sensitive to actinomycin D and it is refractory to rifampicin (32). On DEAE-sephadex it is the first RNA polymerase eluted by a linear ammonium sulfate gradient, generally around 0.2 M (NH4)2SO4. The type I RNA polymerase has a preference for magnesium and low salt concentrations (0.04-0.07 M (NH4)2S04). The type I RNA polymerase is very unstable and therefore difficult to purify (37).







12


The type II RNA polymerase is a nucleoplasmic RNA

polymerase that synthesizes a product which competitively hybridizes with HnRNA (32). Its activity is sensitive to a-amanitin and it is the second RNA polymerase eluted by a linear (NH4)2SO4 gradient from DEAE-sephadex, generally around 0.3 M salt. The type II enzyme exhibits more activity with manganese, rather than with magnesium, and is most active at high salt concentrations (0.9-0.12 M (NH4)2SO4) (32). It is more stable than the type I RNA polymerase, but is inactivated easily, particularly during salt precipitation.

Type III RNA polymerase is a nucleoplasmic RNA

polymerase, eluted third on DEAE-sephadex chromotograph, generally around 0.35 M (NH4)2SO4 (32). The small amount of type III enzyme activity present in extracts is often undetected. The type III RNA polymerase is resistant to a-amanitin, prefers manganese and has a broad salt optimum (0-0.2 M (NH4)2SO4) (32). It is the least studied of the nuclear RNA polymerases and it has been proposed that itmay synthesize tRNA (32).

The type IV RNA polymerase, refers to RNA polymerase activity detected in cellular organelles, specifically chloroplasts and mitochondria. Little is known of these enzymes due to the difficulty in extracting the quantity of material necessary for enzyme purification, and to the difficulty in removing the other contaminating RNA polymerases.







13


Most studies of organelle transcription therefore utilized in vivo: labeling and experimentation.


Structure

RNA polymerase II has been purified from calf thymus, rat liver, sea urchin, yeast, mouse embryo cells, Hela cells, xenopus and maize (1,5,27). The subunit structure of the type II RNA polymerase from calf thymus was evaluated on SDS-polyacrylamide gel electrophoresis (38). These subunits had molecular weights of 215,000, 185,000 and 150,000 and were designated Bl, B2, and B3. Gel densitometry indicated a ratio of 1:1:2 between the gel bands Bl:B2:B3 (39). These subunits apparently came from two subsets of RNA polymerase II. The first subtype gave bands B1 and B3, the second subtype gave bands B2 and B3. There were also 3 smaller subunits of molecular weights 20,000, 30,000 and 40,000. The large subunits Bl, B2 and B3 appeared to be similar to the B and B' subunits of the E. coli RNA polymerase. It has been suggested that o-amanitin inhibits the type II RNA polymerase by binding to subunit B3.


Maize RNA Polymerase

Maize RNA polymerase was one of the first eukaryotic RNA polymerases solubilized (27). Other eukaryotic RNA polymerases were solubilized from animal tissues (1); however, other plant RNA polymerases were studied primarily







14


using plant chromatin or crude salt fractionated supernatants (40,41,42). RNA polymerase activity was first detected in the soluble fraction of a French pressure cell extract in 1964 by Mans and Novelli (43). The RNA polymerase in this extract was further purified by DEAE-cellulose chromotography using a linear Tris-HC1 gradient (Stout and Mans 1967) (27). The average specific activity eluted was 4.06 nmoles AMP/mg at 10 min. This eluted RNA polymerase would utilize either native or denatured DNA as a template equally well, although at low DNA levels (10 Ug DNA/ml) denatured DNA was eight times as effective as native DNA (44). Denatured calf thymus DNA was more efficient as a template than denatured maize DNA; however, the calf thymus DNA had a much greater hyperchromicity than maize DNA, 17 percent vs 8.3 percent (44). The RNA polymerase required all four nucleoside triphosphates, a bivalent metal ion, and DNA to incorporate [8- 14C]ATP into acid-insoluble material (27). The metal could be either magnesium (25 mM) or manganese (5mM). If [a-32p]UTP or [a-32P]ATP were used as labeled substrate, the nearest neighbor frequency indicated RNA containing all four nucleosidemonophosphates had been synthesized (27). The reaction was inhibited by actinomycin D, pyrophosphate and DNase (43). The product synthesized on native DNA was greater than 90 percent digested by pancreatic ribonuclease, while on denatured DNA this decreased to 73 percent (27). On sucrose density gradients the products







15


were 14-16 S, which corresponded to the distribution of the denatured DNA template (44). This distribution was that expected for DNA-RNA hybrids. As expected, upon heating the complexes disaggrated and all nucleic acids were at the top of the gradient (4-6 S).

The RNA polymerase purified by the method of Stout and Mans was identified as a type II nucleoplasmic RNA polymerase by its sensitivity to a-amanitin (45). This type II RNA polymerase did not synthesize homopolymers such as poly(A) with denatured DNA, as many bacterial RNA polymerases did (46), for with denatured DNA "the formation of a homopolymer was not detected with the maize polymerase" (44, p. 752), nor was it detected with any other eukaryotic RNA polymerase.

A type-I maize RNA polymerase was reported by Strain et al. (47). Maize leaves were used as crude material and carried through DEAE-cellulose chromotography. The type I RNA polymerase eluted at 0.08 M (NH4)2s04 (47). Strain, et al. also detected two overlapping peaks of activity in the type II RNA polymerase region.. One peak preferred native DNA, the other preferred denatured DNA as measured by total AMP incorporated (47). The metal requirements of the leaf RNA polymerase indicated a preference for magnesium over manganese, with optimums at 25 mM for magnesium and

8 mM for manganese (47).







16

Polyadenylic Acid


Enzymatic Synthesis

A eukaryotic polyadenylic acid polymerase was first discovered in calf thymus by Edmonds and Abrams in 1962

(48). Its activity was inhibited by the other nucleotide triphosphates, it required magnesium and it was particulate, perhaps bound to the nuclear membrane. Its product was almost pure poly(A), except for about 1 percent of the adenylate residues which were joined to cytidylate residues. The enzyme could not be freed of endogenous RNA. Others have purified poly(A) polymerases from rat liver

(49) and from maize (50). The maize poly(A) polymerase adds poly(A) chains to the 3' hydroxyl of primer nucleic acids, either RNA or DNA (51).

Poly(A) synthesis has also been studied in prokaryotic systems. E. coli RNA polymerase,with denatured calf thymus DNA and only ATP as substrate, will synthesize poly(A) sequences using poly(dT) regions of the DNA as template

(46). Reiteritive transcription of poly(dT) regions, each greater than 5 nucleotidyl residues long, resulted in the synthesis of long poly(A) chains through a repeated utilization of the poly(dT) template by an unknown mechanism. Poly(A) synthesis required denatured DNA and was inhibited by the addition of the other nucleoside triphosphates. The polymerase would not lengthen added poly(A) primers (46).






17



The E. coli RNA polymerase had a greater affinity for denatured DNA than for native DNA (52), perhaps reflecting a greater binding affinity for the exposed poly(dT) regions of the denatured DNA.


Importance

From 1961 to 1968, poly(A) synthesis by prokaryotic RNA polymerase was an unusual artifact of the assay and of unknown significance. With the discovery of poly(A) se.quences in RNA isolated from numerous eukaryotes; vaccinia virus cores (53), Hela cells (54,55), mouse sarcoma cells

(56), and avian myeloblastosis virus (57), poly(A) synthesis again became of interest. In eukaryotes only a portion of the DNA-like nuclear RNA is transported to the cytoplasmic polysomes. In addition, the nuclear RNA is much larger in size than that found in the cytoplasm (53). In studying poly(A) synthesis in vaccinia viral cores, Kates asked, "Could poly(A) sequences in nuclear RNA play a role in either the cleavage of RNA into smaller pieces or in the selective transport of certain species to the cytoplasm?" (53, p. 752). To answer the question of the role of poly(A), two additional questions should first be answered. Is the poly(A) covalently attached to the RNA, and if so, where? If attached to RNA, what enzyme catalyzed the synthesis of the poly(A) sequence? Finding some poly(A) attached to RNA does not imply that






18



all the poly(A) was attached, or remained attached. Knowing the time and location of poly(A) synthesis and the enzymes responsible would contribute significantly to an understanding of the physiological function of poly(A) synthesis.



Location in Vivo

Poly(A) can be isolated from the HnRNA or from the

rapidly labeled RNA isolated from polyribosomes (54,55,56). Edmonds (54) indicated the data was consistent with the idea that every HnRNA contained at least one poly(A) sequence. More than one mode of poly(A) synthesis was implicated since cordycepin (3-deoxyadenosine) suppressed the labeling of mRNA found on Hela ribosomes while not effecting the labeling of nuclear RNA (58). This suggested there may be two enzymatic activities that synthesized poly(A), one sensitive to cordycepin that synthesized poly(A) and one insensitive to cordycepin that synthesized HnRNA containing poly(A) sequences. Lim and Cannelakis'

(59) results with haemoglobin mRNA-indicated that, at most, it could contain 70 polypurine residues of 70 percent AMP. Furthermore, this polypurine sequence was not at the 3' end of the haemoglobin mRNA, since the 3' end contained only 7 or 8 AMP residues before the first pyrimidine (60). Therefore, the poly(A) must be at the 5' end or inside the RNA chain, if there at all.







19


ie strongest evidence for poly(A) being contained

in newlk ,synthesized RNA was that of Kates (53). Utilizing

-vaccimiia scores incubated in vitro, under conditions of RNA synthesis, Kates' data indicated that after mild RNase treatment all of the RNase resistant poly(A) sequences sedimentated at 4 S and had a chain length greater than 50 nucleotides. The association of poly(A) with RNA was not disrupted by heating at 100', nor by 75 percent DMSO at 800 in the presence of cold poly(A). This indicated that, if in fact, the poly(A) was attached to the RNA it was probably through a covalent bond. When poly(A) was synthesized by vaccinia cores with only ATP as the substrate, then the poly(A) was not later attached to newly synthesized viral RNA (53). This in vitro poly(A) synthesis continued for only 5 min.,.the poly(A) was 180 nucleotides long and had a uniform 5.8 S value. After alkaline hydrolysis and chromatography there was one 5' tetraphosphate and one adenosine for every 180 AMP residues (53). If synthesis ,o=urred in the presence of all 4 nucleoside triphosphates, 25-30 percent of the AMP incorporated was in poly(A). The poly(A) was synthesized without a lag period upon addition of the substrates, but if UMP incorporation was measured, there was a 1.5 min. lag before incorporation began (53). This indicated poly(A) synthesis preceded RNA synthesis. If the poly(A) synthesized in the presence of all 4 nucleoside triphosphates was purified and alkaline hydrolyzed,








20


there were 5 adenosine residues for every adenosine tetraphosphate (53). Therefore, if poly(A) was the initial sequence and contained the tetraphosphate, there were four more sequences that were synthesized internally or at the 3' terminis.

Based upon hybridization of poly(A) to denatured

vaccinia DNA, Kates estimated there could be greater than 25 poly(dT) sequences in the vaccinia DNA, each 180 nucleotides long (53). This wag consistent with Heaust and Botchan (61), who stated that 10 percent of the genome of mice consisted of AT-rich regions. The function of this DNA was not clear, but it was probably not transcribed in vivo (61). It was, however, known to be uniformly distributed among all chromosomes.


Summary

Poly(A) sequences are known to occur in vivo and in

vitro. They appear to be synthesized by two enzyme systems, poly(A) polymerases sensitive to cordycepin, and by RNA polymerases insensitive to cordycepin. Some poly(A) sequences are located at the 3' hydroxyl end of RNA polymers' however, there is evidence for both poly(A) sequences internally and at the 5' end of the RNA. The importance and functional significance of these sequences is not firmly established.














METHODS AND MATERIALS



Methods


RNA Polymerase Purification

The purification of maize RNA polymerase has evolved

through enumerable modifications of that published initially

(27). Each step in the procedure utilized here is indicated in Figure 1.


Step 1. Preparation of material

Grain (4-10 liters), in a plastic garbage can with a

perforated bottom, was imbibed and germinated under running water (230) for five days until the shoots were 2 to

4 cm long. The roots and shoots were separated from their kernels with a vibrating, stainless steel gravel separator under a shower, collected batchwise in a strainer, excess water was shaken out, and then the material was dropped into liquid nitrogen. Packets of frozen shoots and Iroots (90 g each) were wrapped in aluminum foil and stored in a Revco freezer (-76*).


Step 2. Homogenization

One packet of seedling tissue was homogenized in a Waring blender in 135 ml of Buffer H for 60 sec. at low 21




22


SEEDLINGS
STEP I SEPARATE

I KERNELS ROOTS AND SHOOTS

STEP 2 HOMOGENIZE

FILTER BOUND FILTRATE
STEP 5 CENTRIFUGE

PELLET SUPERNATANT
-.4T
STEP 4 50% (NH4)2S04

SUPERNATANT PELLETSTEP 5 SEPHADEX G50 INCLUDED VOLUE EXCLUDED VOLUME
I
STEP 6 DEAE-CELLULOSE

FLOW THROUGH I RNA POLYMERASE


Figure 1. PURIFICATION PROCEDURE FOR RNA POLYMERASE







23


speed and for 15 sec. at high speed. The homogenized material had the consistency of a thick milkshake. The homogenized material was immediately filtered through four layers of cheesecloth, through a layer of miracloth and into a chilled Erlenmeyer flask. Material retained in the filters was discarded.


Step 3. High speed centrifugation

The filtered homogenate containing 20 to 40 percent of the protein present in the shoots and roots was centrifuged for 60 min. in a Ti50 rotor at 200,000 x g at 0*. The supernatant fraction was decanted into a chilled graduated cylinder through a layer of miracloth, which retained the lipid layer accumulated at the top of the centrifuge tube. The supernatant fraction contained approximately 50 percent of the protein present in the filtered homogenate. Step 4. Ammonium sulfate precipitation

To the high speed supernatant an equal volume of

saturated (NH 4)2 so 4 was added slowly with continuous and IV

gentle stirring in an ice-j acketed beaker. After 30 min. of stirring the resulting precipitate was collected by centrifugation (10,000 x g, 10 min., 01) and resuspended in a minimal volume of Buffer R (approximately 15 ml).







24

Step 5. Salt equilibration on sephadex

..Equilibration in Buffer E was accomplished by passage of the resuspended precipitate through a sephadex G50 uolumn (2.5 x 11 cm) equilibrated with Buffer E. The excluded material was diluted to 25 ml with Buffer E for absorption to DEAE-cellulose. Step 6. DEAE-cellulose chromotography

The excluded volume eluted from sephadex G50

chromotography was loaded (1 ml/min.) onto a DEAE-cellulose (see materials) column (2.5 i.d. x 11- cm) equilibrated with Buffer E. The loaded column was washed with 60 ml of Buffer E and the flow rate was decreased to 0.25 ml/min. A 60 ml (NH4)2S04 gradient (0.20 to 1.0 M) was begun immediately after passage of the 60 ml of Buffer E. Column eluates were monitored and recorded by a continuous flow ultraviolet-monitoring system (Gilford spectrophotometer and a Honeywell recorder, Figure 2). Eluted fractions were collected directly from the flowcell into glass vials (2.5 ml/vial)and frozen in liquid nitrogen. Polymerase Assay

RNA polymerase was assayed in a 0.10 ml standard

reaction mixture containing: 10 pmoles Tris-Hcl, pH 7.6 @ 250; 0.25 pmoles each UTP, CTP, GTP, (sodium salts);

0.10 Pmoles [8-14C]ATP (specific activity 1.7 to 4.5 vC/pmole)

1.0 mole MgC12; 1.0 mole 2-mercaptoethanol; 8 moles






25

z
w
Cl) C, CD
w oso

> M




SPECTRE
O.j
I -iw 0
I PHOTOMETER U
w






DEAE COLUMN
0- to

0. 0
4
w GRADUATED CYL.1
CL 1 C4

ri


C
$4
CS]
co P4
z z w 2



0







26


(NH 4)2 so4; 0.37 A20calf thymus DNA; 5 iig BSA and enzyme

as indicated (27). An assay mixture was prepared immediately before use and held on ice until the reaction was initiated by the addition of enzyme to the mixture. The reaction tube was immersed in a 300 water bath for 20 min. and acidinsoluble radioactivity was determined by a modification of a procedure previously described (27). The reaction was terminated by pipetting the mixture onto a 3 MM filter paper disk. The reaction mixture was absorbed into the disk for 10 sec. under a heat lamp and precipitated by immersion in cold 10 percent TCA containing 2 mM sodium pyrophosphate. As many as 40 disks were then extracted five times with 100 ml of the TCA solution (5 min. each time), once with 100 mlethanol-ether (1:1), and once with 100 ml ether for 3 to 5 min. All extractions were performed at room temperature. The disks were then dried and counted in a scintillation solution containing PPO, POPOP and reagent grade toluene. Disks which received reaction mixture containing no enzyme averaged 15 cpm above machine background. The wash procedure reduced the cpm from approximately 400,.000 cpm/disk to 35 cpm/disk for the no enzyme control. Machine counting efficiency was 0.75 cpm/dpm. One unit of RNA polymerase activity is defined a s 1 nmole AMP incorporated into acid-insoluble material under standard reaction mixture conditions in 20 min.







27

Protein Determination

Protein was measured on samples precipitated with 10 percent trichloroacetic acid by the method of Lowry et al. (62), using BSA as a standard. Ribonuclease Treatment

Pancreatic ribonuclease (80 pg/ml), heated at 80*

for 10 min. to inactivate DNase, was added to 0.1 ml aliquots of an incubated reaction mixture to a final concentration of 20 pg/ml, and incubated for an additional 10 min. at 301 (54). Acid-insoluble material remaining was determined on filter paper disks with the polymerase assay wash procedure (see polymerase assay).


Alkaline Hydrolysis

Product, isolated by sephadex gel filtration or by KC104 precipitation from an incubated reaction mixture, was suspended in 1.0 ml of 0.3 M KOH and incubated at 37' for 18 h in a stoppered test tube. After the 18 h hydrolysis, the solution was acidified with 70 percent HC104 and the precipitate removed by centrifugation. The supernatant was neutralized with 0.3 M KOH and the salt was removed from the hydrolysate by charcoal adsorption (63). The charcoal column was washed successively with water ( 3 ml) and 0.1 M NH4OH (5 ml) and the nucleotides eluted with 5 ml of ethanol-NH4OH (ethanol-conc NH4OH-H20, 2:1:2 by volume). The eluted fraction was lypholyzed to dryness and resuspended in water.







28


Paper Electrophoresis

Samples to be electrophoresised were adsorbed to

paper strips (4 x 30 cm, Whatman #1) by repeatedly applying and drying on one spot (5 to 10 mm dia). The paper strips were then subjected to electrophoresis in 0.025 M sodium citrate, pH 3.5, for 4 h at 300 v, 6-8 amps, at 251 in a Universal Electrophoresis Cell (Buchler Instruments).


Polyacrylamide Gel Electrophoresis

Polyacrylamide gels for native proteins were prepared and run according to the method of Davis (64) using a stacking gel and 5.2 percent polyacrylamide gels. The gels were run at pH 8.8 and stained with aniline blue black. The 10 percent polyacrylamide gels and denatured enzyme protein were prepared and run according to the method of Weber and Osborn (65). The 10 percent gels were run at pH 7.0 in 0.1 percent SDS and stained with coonassie brilliant blue.





Materials


DEAE-cellulose, Bio-RAD, Cellex-D (6.1 meq per g,

dry weight), was washed by the method of Peterson and Sober

(66), equilibrated in 0.05 M Tris-HCl, pH 7.6 at 250, and stored at 40. Sephadex G50, medium grade, Pharmacia, was







29


swollan..and e.q~i.ibrated in 0.05 M Tris-HCl, pH 7.6 at 250, and ~tored:-D:ovine serum albumin, 5X crystallized, was,, .from Pen a~iochemicals. The nucleoside triphosphates GTP, CTP, UTP -and ATP were purchased as sodium salts from Schwarz/Mann, including [8-14C]ATP, [2-14C]UTP and [a-32p] ATP. Calf thymus DNA was purchased from Schwartz/Mann, and pancreatic ribonuclease, chromatographically pure, was purchased from Worthington Biochemicals. Actinomycin D was purchased ffrm Schwarz/Mann and cordycepin, grade C, was purchased from Sigma. Alpha-amanitin was a gift of Dr. T. Weiland and rifampicin was a gift from GruppoLepetit S.P.A. Research Laboratory. Zea mays L., WF9 x Bear 38, waxy, was purchased from the Bear Hybrid Seed Co. Reagents

Calf thymus DNA was dissolved in 0.1 x SSC at 37 A26/ ml and stored in 0.2 ml aliquots at -170 (67). An aliquot was thawed for use before each assay. Denatured DNA was prepared by the dilution of a freshly thawed aliquot of DNA in 0.1 x SSC (1:1 v/v) into a sealed vial, 5 min. immersion in boiling water, followed by quick chilling in iced-water (average hyperchromicity 24%). All the A260 measurements in the tables and figures represent the absorbancy before denaturation.

Stock solutions of 1.0 M Tris-HCl, pH 8.0 or pH 7.6 at 250, 1.0 M MgCI2, 14.7 M 2-mercaptoethanol were used to make the following buffers: Buffer H, 0.25 M sucrose,







30


0.10 M Tris-HCl, pH 8.0, 1.0 mM MgCl2, and 50 mM 2-mercaptoethanol; Buffer E, 0.05 M Tris-HCl, pH 7.6, 10 mM 2-mercaptoethanol and 0.20 M (NH4)2S04; Buffer R, 0.05 M Tris-HC1, pH 7.6, 10 mM 2-mercaptoethanol. Buffers were made immediately before use, using warm glass-distilled water to minimize gas bubbles in the column eluates passing through the spectrophotometer flowcell.

Saturated ammonium sulfate was kept at 40 with

crystals visible in the bottom of the bottle. Before use in protein precipitations, concentrated NH4 OH was added drop-wise until a 1:20 dilution of the (NH4)2S04 was pH 8 at 250 (67). Sodium chloride-sodium citrate buffer (SSC)

1 X, was 0.15 M NaCl, 0.015 M sodium citrate, pH 7.3 at

250 (68).













RESULTS



RNA Polymerase Purification


Utilizing the RNA polymerase .purification procedure described in METHODS, RNA polymerase was purified 200-fold over the 200,000 x g supernatant activity, or 1,000-fold over that present in the tissue homogenate. The results of several RNA polymerase purifications are summarized in Table 1. The steps in purification correspond to those in Figure 1. In 10 h, 2 mg of RNA polymerase were purified from a 90 g packet of maize roots and shoots, with a recovery from 55 percent to 69 percent of the initial AMP incorporating activity present in the 200,000 x g supernatant. Variability in protein and activity of the 200,000 x g supernatant probably resulted from variability in homogenization with the Waring blender. Activity detected in the 50 percent (NH4)2SO4 precipitate was variable (Table 1), resulting from the presence of nucleases and variable amounts of salt in the resuspended protein. After salt equilibration on sephadex G50, assays of polymerase activity were less variable.

Both gradient and batch elution of DEAE-cellulose

resulted in highly active RNA polymerase. Gradient-eluted RNA polymerase had a higher peak specific activity (104nmoles 31







32



TABLE 1

STEPS IN RNA POLYMERASE PURIFICATION



A standard reaction mixture contained 0.37 A260 DNA, 1.0 uimole 260
MgCl2, and enzyme..as follows: 96 uig 200,000 x g supernatant, 500 pg 50% ASP, 250 ug sephadex G50, or 3 to 5 pg DEAEcellulose fraction.


Protein Specific Total Yield Step Activity Activity


(mg) (nmoles AMP/mg)(units) (%) 200,000 x g Supernatanta 507+100 0.570.12 29090 100

50% ASPa 207 50 0.720.3 19560 6730
Sephadex G50a 250 50 0.700.07 17520 6010

DEAE-celluloseb
Gradient elutionc 2.0 66.0 132 55

Batch elutiond 5.0 40.0 200 69




aAverage of 3 preparations. bFlowthrough contained less than 5% of added RNA polymerase
activity.

CGradient elution, peak tube specific activity, 10:4 tnmoles
AMP/mg; protein, 0.2 mg/ml.

dBatch elution (0.4 M (NH4)2SO A), peak tube.specific activity,
90 nmoles AMP/mg protein 0. mg/m1.







33


AMP/mg), however, it was more dilute (0.2 mg/ml) and its preparation more time consuming (6 h). Batch eluted RNA polymerase had a lower peak specific activity C90 nmoles AmP/ mg), but it was more concentrated (0.5 mg/ml), more rapidly eluted (2 h) and the net recovery was better than with the gradient procedure.

The profile of A280 material during gradient elution of the DEAE-cellulose column indicated most of the material failed to bind to the resin equilibrated with 0.2 M (NH4)2 S04(Figure 3). However, all of the RNA polymerase activity was bound to the resin. Of the two partially resolved peaks of A280 absorbing material eluted from the resin by the (NH 4)2So4 gradient, shown in more detail in Figure 4, only the second peak exhibited RNA polymerase activity. Although the RNA polymerase activity detected with denatured DNA was twice that detected with native DNA, the ratio of specific activities with native and denatured DNA was constant among the eluted fractions of the second peak. Attempts to enhance purification by equilibration of the resin with higher than 0.2 M (NH4)2So4 to reduce binding of the protein in the first peak (Figure 4) were unsuccessful. If the salt concentration was increased to 0.21 M, 15 percent of the enzyme activity appeared in the column flowthrough.







34

















040

E




C4




14 IV 3 N C201 0S G0












1.0 U -0.













0 0. 41, 0.1
E 0




2 O 250 24






VOLUME m!




Figure 4 RNA POLY-MERASE ACTIVITY IN
DEAE-CELLULOSE FRACTIONS

Assayed in a standard reaction mixture
containing 0.5 moles MnCl2, 0.037 A260 DNA; either denatured (solid triangles)
Cs.
























or native (open triangles) and to 4 g
enzyme.
20 230 240

VOLUME ml





Figure 4. RNA POLYMERASE ACTIVITY IN
DEAE-CELLULOSE FRACTIONS
Assayed in a standard reaction mixture containing 0.5 moles MnCI2, 0.037 A260 DNA; either denatured (solid triangles) or native (open triangles) and to 4 iig
enzyme.







36


Polyacrylamide Gel Electrophoresis

Awaliiquot of the proteins showing the highest RNA

-polymera~e specific activity (eluted from DEAE-cellulose at 235 wi in Figure 4) was examined by electrophoresis on 5.2 percent polyacrylamide gel. Several components were visible, with more than 10 bands staining with aniline blue black (Figure 5). All the protein staining material migrated into the gel, toward the anode at pH 8.8. Therefore, the proteins are acidic and none were excluded by the 5.2 percent polyacrylamide gel. The slowest migrating and darkest staining band, however, may represent an aggregate of some of the more rapidly moving components. The intensity of this major band was approximately equal to that of the BSA standard (20 ig) and, therefore, may account for 50 percent of the added protein. If an aliquot of the same protein preparation was denatured with 0.1 M 2-mercaptoethanol and 0.1 percent SDS, then subjected to electrophoresis on 10 percent polyacrylamide-0.1 percent SDS gels, the number of visible bands was reduced to 5 (Figure 6). Two distinct bands, migrating 13 and 14 mm into the gel, a faint band at 19 mm znd a still fainter pair of thin bands at 21 and 23 mpm were characteristic of all the purified maize RNA polymerase preparations. The decrease in detectable bands on SDS-polyacrylamide as compared to the 5.2 percent polyacrylamide gels may reflect disaggregation of the denatured proteins. Alternatively, the presence of more







37


















!I
3. I


















_ A (+) B

Figure 5. POLYACRYLAMIDE GEL ELECTROPHORESIS OF
NATIVE RNA POLYMERASE

Samples were run on 5.2% polyacrylamide gels at pH 8.8 (see METHODS). Gel A contained 40 ig peak RNA polymerase (eluted at 235 ml, Figure 4). Gel B contained 20 ig BSA.







38

(-)
































A B C
(+)




Figure 6. POLYACRYLAMIDE GEL ELECTROPHORESIS OF
DENATURED RNA POLYMERASE

Samples were denatured in 0.1% SDS, 0.14 M 2-mercaptoethanol at 370 and run on 10% polyacrylamide gels containing 0.1% SDS at pH 7.0 (see METHODS). Gel A contained 20 pig peak RNA polymerase (eluted at 235 ml, Figure 4). Gel B contained 6.2 ig denatured BSA. Gel C contained denaturing buffer only.







39


bands on the gel run with native protein may reflect greater senslti ity of band detection since the sample analyzed con-tained :bwice as much protein and the gel was stained with anilineblue black rather than coomassie brilliant blue. Assuming that the distance migrated by the denatured protein components in SDS-polyacrylamide gels was a function of their molecular weights, then the pair of bands migrating 21 and 23 mm (equivalent to the migration of BSA) were about 65,000 to 75,000 d. Therefore, the other bands characteristic of the RNA polymerase preparations corresponded to polypeptides of higher molecular weight. Enzyme Stability

Storage temperature and freeze-thaw

Maize RNA polymerase collected in glass vials and immediately stored in liquid nitrogen lost no activity after 3 months. The enzyme was stable for 19 days at -76*, but lost all activity at -171 (Table 2, part A). The addition of glycerol to 20 percent by volume to fresh enzyme preparations did not stabilize the polymerase enough to make storage at "170 practical. However, in 20 percent glycerol the enzyme did retain 50 percent activity for 17 h at 40 (Table 2, part B). The enzyme stored in liquid nitrogen was stable to repeated freeze-thaw. The loss of activity observed may reflect decay accumulated during the time the enzyme was held on ice preceding assay (Table 2, part C).







40


TABLE 2

STORAGE AND FREEZE-THAW LABILITY OF RNA POLYMERASE



After the indicated treatment aliquots of one enzyme preparation were assayed for RNA polymerase activity in a standard reaction mixture containing 1.0 mole MgCI2,
0.37 A260 native DNA and 18 jig enzyme. 100% incorporation' was equivalent to 172 pmoles AMP incorporated per reaction.


% Incorporated

A) Temperature (19 days)

-761 (Revco freezer) 98
-170 (Freezer) 1

B) Glycerol Storagea (17 h)

-196* (Liquid nitrogen) 96
-17 (Freezer) 78
40 (Refrigerator) 48


C) Freeze-thawb

One 100
Two 100
Three 86
Four 82
Five 79
Storage on Ice (5 h)c 80


aGlycerol was added to 20% by volume to the enzyme preparation
just prior to storage.
bA vial of enzyme frozen in liquid nitrogen was thawed at 370
and held on ice prior to assay. After assay it was rerozen
in liquid nitrogen. This sequence was repeated as indicated. CEnzyme frozen and thawed once prior to 5 h storage on ice.







41

Salt precipitation

In :.an aimpt to concentrate the RNA polymerase eluted ,from DEAE-:ceJlLlose, solid (NH4)2So04 was added to 80 percent saturation to the pooled fractions. Only 10 to 15 percent of the eluted activity was recovered in the precipitated protein. To facilitate precipitation, calf thymus DNA was added in varying amounts to the pooled fractions just before salt addition. Addition of DNA resulted in an increase in precipitated activity, roughly porportional to the amount of DNA added (Table 3). Low recovery of polymerase in precipitates from a solution with high DNA levels (300 pg/ ml) suggested formation of a DNA-protein complex soluble in high salt. Neither polymerase activity in the supernatants nor protein in the precipitates was determined, therefore inactivation of polymerase and specific activity were not assayed per se.


Summary

DNA-dependent RNA polymerase was purified 1,000-fold from an homogenate of maize shoots and roots. Following DEAE-cellulose chromotography the enzyme specific activity was greater than 100 nmoles AMP incorporated/mg enzyme at 20 min. The recovery was greater than 50 percent of the activity detected in the high speed supernatant. Polyacrylamide gel electrophoresis of both native and SDSdenatured proteins indicated the RNA polymerase preparation still contained several polypeptides. Nevertheless the







42

TABLE 3

SALT PRECIPITATION OF RNA POLYMERASE




Each sample contained 0.75 mg protein before 80% saturation with solid, powdered (NH4)2So4. Just before addition of salt, DNA was added as indicated, then salt was slowly added while the mixture was stirred on ice for 15 min. The precipitate was collected by centrifugation (10,000 x g for 10 min.), resuspended and desalted in Buffer R on sephadex G25. Samples were assayed in a standard reaction mixture
-with 1.0 mole MgCl2 and 0.37 A260 DNA.



DNA Added (jig/ml) % Activity Recovered a



None 13
2.4 20
6.0 30
12.0 38
30.0 46
300.0b 10



control incorporated 185 pmoles AMP.

very little precipitate obtained.





dt







43


denatured protein exhibited those bands characteristic of eukaryoti'c RK.'polymerases. The enzyme was stable after freezing anti~sturage at -761. Precipitation of the purified enzyme with"'(NH4) 2S04 was facilitated by DNA. Further resolution of the proteins in the RNA polymerase preparation was thwarted by the instability of the enzyme at 40 and by the lack of recovery of active enzyme following salt precipitation.





RNA Synthesis


Assay Requirements

Enzyme purified by the procedure described in METHODS exhibited the expected requirements for RNA synthesis. DNA, metal and all four ribonucleosidetriphosphates were required for incorporation of labeled AMP into acid-insoluble material (Table 4). Denatured DNA was utilized as well as native calf thymus DNA, and maize DNA (not shown here) supported comparable activity. Mn2+ satisfied the metal requirement as well as Mg 2+, in fact, better than Mg2+ when denatured DNA was provided as the nucleic acid component. Incorporation of AMP in the absence of NTPs was significant, especially when assayed in the presence of denatured DNA. All of the radioactivity detected in acid-insoluble material on filter paper disks was dependent upon the addition of enzyme protein.







44






TABLE 4

ASSAY REQUIREMENTS FOR MAIZE RNA POLYMERASE




A standard reaction mixture contained 0.37 A260 DNA, 1.0 mole 260
MgC12 or 0.5 moles MnC12 and 5 jg RNA polymerase.




System AMP Incorporateda (pmoles)
Native DNA Denatured DNA


Complete 295 327

Complete less DNA 0 1

Complete less Mg2+ 0 1

Complete less Mg2+, plus Mn2+ 300 551

Complete less NTPs 10 58

Complete less enzyme 0 0



aTotal radioactive AMP incorporated per reaction mixture.


Es







45


DNA
... 2+
1.n 'e' Iete system with Mg as the metal cofactor,

-the 1',Aw require-ient was satisfied with either native or heat-denatured'"ONA (Table 4). The reaction was saturated with denatured DNA in excess of 0.4 A260/ml, whereas with native DNA more than a 5-fold higher concentration (1.8 A26& ml) was required to reach the same level of activity (Figure 7). If equal amounts of native and denatured DNA were combined and !assayed in the same reaction mixture, the activity measured could be accounted for by the denatured DNA alone. Thus, the denatured DNA appeared to out-compete the native DNA .fbr the RNA polymerase. However, at saturating concentrations native and denatured DNA supported equivalent AMP incorporation.

If MnCl2 was substituted for MgCl2 in the reaction

mixture, there was almost a 2-fold increase in RNA polymerase activity with denatured DNA (Table 4). In the presence of Mn2+, the activity with denatured DNA was twice that with native DNA over a range of DNA concentrations (Figure 8). Although the RNA polymerase assay was saturated at 0.4 A260/ ml with both native and denatured DNA, AMP incorporation after 20 min. was not equivalent. Therefore, the concentration dependence of the polymerase on native and denatured DNA differed when assayed with Mg 2+, but the maximum level 2+
of activitywas- the same. In contrast with Mn the concentration dependence with native and denatured DNA was








46








En

U 04
0
C*j -H

;:L 4
0 r-q -P 0 X 0 r-4 M -H
tm
r a) ro rT4 > 0
c -H 4
u -H 4J :J
fd (d 4-) 4-)
0
0 rO ro
(1) -P a)
>
:Z 4-J -H 4J
x ro rd
r4
z ro
0 U) r. E-4 0 ro m
4.)
40
0 k ra r-I
1-4 9 tm
P ro m
4
E-4 rC$ U) 4-) H r (a
E-4 fd k rd
-P a) -rq fc
>-, 0
r-i En 0
Z Q4 ra
Q)
ro
a)
>1

U) Q)
F: rO


cli


S-31OWd G31.:MOdE3-ONl dN V








47









0 ro

'H
U) r-4 Q) 0
-1 rn 0

>
Ln -ri P4 -P 0 C) m


rZ4 -H rd
0
u
(d (d

0 rcj
E
W Q) En Z 4 0 4 j %0 0 -P b-I Z X 9 r


W r, rd 4-)
*now E-q 0 1:1
ro ro :it 4-) -,1
() (1) r-i
z cn, M U) 0
Z 0 ro m
0 4 4
E-4 rZ:$ F
4 >i C) Q$ r-i 4 p ro 0 5 H r 04 4-) P rt 4-)
4 ul Z (1) z P ro
Q rd
z 0

ro M
>1 CN r-I
4





salowd (131VNOdUO.4jNNI dWV







48


similar but the maximum level of AMP incorporation differed.


Metal

For maize RNA polymerase activity there was an

absolute requirement for a metal ion cofactor satisfied
2+ 2+
with either Mg or Mn (Table 4) 4 There was a broad
2+
Mg optimum concentration from 10 to 14 mM, centered at 12 mM, and independent of the nativity of the DNA (Figure 9). Routinely, however, 10 to 25 percent more activity was observed with denatured DNA than with native DNA. The 2+
maximum AMP incorporation occurred with 5 mM Mn2, either with native or denatured DNA (Figure 10). With denatured DNA, the RNA polymerase activity was twice that with native
2+
DNA at all 2Mn levels. As is characteristic of polymerases,
2+
the Mn optimum concentration was sharper than that of
2+
Mg.


Substrate

With native DNA, 96 percent of the AMP incorporation required the presence of the NTPs (Table 4). The RNA polymerase reaction was saturated with the pooled NTPs above

1.0 mM for each of the three nucleoside triphosphates; GTP, CTP and UTP and no nucleotide inhibition was detected with a 5-fold excess (Figure 11). The standard reaction was 2.5 mM with each NTP; thus the assay of polymerase was not limited with respect to unlabeled nucleosidetriphosphates.

The RNA polymerase reaction mixture was saturated







49







CO






0
E

400








0201
z



ME/



0 10 20 40

MAGNESIUM CHLORIDE mM








Figure 9. MAGNESIUM TITRATION WITH
NATIVE AND DENATURED DNA

Assayed in a standard reaction mixture containing 0.37 A260 DNA; native (solid circles) or denatured (solid triangles),
6.4 uig RNA polymerase and MgCl2 as
indicated.






50






600






UJ 4001
0
I-.



0
OO


0









0 o0 20

MANGANESE CHLORIDE WM



Figure 10. MANGANESE TITRATION WITH
NATIVE AND DENATURED DNA

Assayed in a standard reaction mixture containing 0.37 A260 DNA; native (solid circles) or denatured (solid triangles),
6.4 pg RNA polymerase and MnCl2 as indicated.






51








o







0

o 200
0
I-L









0 0.5 ID 1.5 2.5 5.0

NTPs mM








Figure 11. NTP TITRATION OF RNA POLYMERASE Assayed in a standard reaction mixture containing 1.0 'moles MgCl2, 0.37 A26 native
oZ 2O











DNA, 6.4 pg RNA polymerase and pooled NTPs
(GTP, CTP, UTP) as indicated.







52


with ATP above 1.0 mM (Figure 12). A Lineweaver-Burke reciprocal plot resulted in a Km of 1.25 x 10 M ATP ( figure 13). The Km was the same with native and denatured DNA, although the Vmax with denatured DNA was twice that with native DNA. The standard ATP concentration for the RNA polymerase reaction mixture was 1.0 mM, since it resulted in the highest ATP specific activity while maintaining the maximum rate of RNA synthesis. The rate of AMP incorporation decreased when the ATP concentration was below saturation. If the ATP concentration was decreased to 0.5 mM there was 86 percent of the maximum rate and if it was decreased to 0.25 mM there was 68 percent of the maximum rate. The pmoles of AMP incorporated per reaction was independent of the [8-14C]ATP specific activity (see APPENDIX A.).


Salt

As with the partially purified enzyme, the more highly resolved preparations exhibited a dependency on added (NH 4)2S04 for RNA polymerase activity, especially in the presence of native DNA (Figure 14). The maximum level of activity occurred between 60 and 90 mM (NH4)2SO4 with native DNA, and up to 100 mM with denatured DNA. Because the RNA polymerase fraction contained 0.4 M (NH4)2S04, the titrations at concentrations below 40 mM were not performed.






53





O



:0
0 4 0 0 ..........






I 200
0
o /
z


0
a./


0 10. 2.0 4.0
ATP mM










Figure 12. ATP TITRATION OF RNA POLYMERASE

Assayed in a standard reaction mixture containing
1.0 mole MgC1 0.37 A2 native DNA, 5 pg RNA
polymerase and ATP as ingIcated.






54


















V

&





/
100








0 4 8

I

ATP


Figure 13. LINEWEAVER-BURKE PLOTS OF ATP
TITRATIONS

Assayed in a standard reaction mixture containing 0.5 Umoles MnCl2, 0.37 A26 DNA, either native (solid circles) or denatured (solid triangles), and 4 yg RNA polymerase. The ATP concentration was mM with the K at 0.125 mM. The V was 100 moles AMP/mg/28 min. for denaturmXDNA and 50 moles AMP/mg/20 min. for native DNA.






55







"540O
E



- 4 0


a: 200o
0
0. 1








0 100 200
AMMONIUM SULFATE mM




Figure 14. AMMONIUM SULFATE TITRATION OF
RNA POLYMERASE
Assayed in standard reaction mixture containing 1.0 pmole MgCl2, 0.37 A260 DNA,
either native (solid circles) or denatured
(solid triangles), 6.4 pg RNA polymerase and
(NH4)2SO4 as indicated.







56


Enzyme

If enzyme protein was not added there was no AMP incorporation in the standard RNA polymerase reaction mixture (Table 4). The total AMP incorporated in 20 min. was the function of the enzyme protein added, up to 6 jig protein per standard reaction mixture .(Figure 15). In the presence of the NTPs, the amount of AMP incorporated with denatured DNA was twice that incorporated with native DNA, at all enzyme concentrations. Rate of AMP IncorToration

The amount of AMP incorporated in a standard RNA polymerase-reaction increased as a function of time for more than 90 min. (Figure 16). The rate of AMP incorporation decreased continuously during the initial 20 min. of incubation and then became constant. The initial rate

(R1) and the late rate (R2) were arbitrarily defined as that occurring up to 20 min., and that occurring after 20 min. What appeared to be two different reactions (initial and late) were not resolved by increasing the incubation temperature from 30' to 37*. Nearest Neighbor Frequency

Product synthesized on denatured calf DNA with

[a-32p]ATP after alkaline hydrolysis, yielded four labeled nucleotides (Table 5). Since the product was acid-insoluble and excluded from sephadex G25, indicating an oligomer, the nearest neighbor frequency data indicated the product







57










80

E







0


0









0 2 4 6

PROTEIN pg



Figure 15. RNA POLYMERASE ACTIVITY AS A FUNCTION OF ENZYME CONCENTRATION

Assayed in a standard reaction mixture containing 0.5 moles MnCI2, 0.37 A260 DNA, either native (solid circles) or denatured (solid triangles), and enzyme protein as indicated.






58

600r






o 00
O
C


i

,o .00CL
0

z
L%




OCA
0 50 100
TIME min






Figure 16. RATE OF AMP INCORPORATION AS A
FUNCTION OF TIME
Assayed in a standard reaction mixture (7X) each containing 1.0 pmole MgCl2, 0.37 A260 native DNA
and 18 pg RNA polymerase. The reaZLtions were incubated at either 30' (solid circles) or 370
(solid triangles) and 0.1 ml aliquots removed at
the times indicated.







59





TABLE 5

NEAREST NEIGHBOR FREQUENCY ANALYSIS OF PRODUCT
SYNTHESIZED WITH DENATURED DNA




A standard reaction mixture containing 0.37 A260 denatured DNA, 0.10 moles [a-32p]ATP (specific activity 30 mCi/mmole),
0.4 moles MnCI2 and 10 jg enzyme was incubated at 300 for 60 min. 1% SDS was added to the reaction, it was passed over sephadex G25, the material in the excluded volume was hydrolyzed in 0.3 M KOH for 17 h at 370, adsorbed to activated charcoal and the eluted nucleotides lyophilized, resuspended in H2 0 and resolved by paper electrophoresis. (see METHODS).



CPM CpA ApA GpA UpA

Added Recovereda



55,000 27,600 0.25 0.44 0.18 0.13




aSome of the sample was lost during lyophilization.






60


was RNA. The relatively high ApA frequency derived from product synthesized with denatured DNA suggested the .presence of poly(A) regions in the product.


Summary

The maize RNA polymerase required added DNA, the

four nucleosidetriphosphates; ATP, GTP, CTP and UTP, and 2+ 2+
a metal, either Mg or Mn for activity. The maximum activities with native and denatured DNA were equal when
2+
Mg was a cofactor. The maximum activity with denatured DNA was twice that with native DNA if Mn2+ was the metal cofactor. Enzyme activity was salt-dependent and proportional to the amount of enzyme protein added. The substrate reached saturation above 1.0 mM and the reaction continued for at least 90 min. The product with denatured DNA had a high ApA nearest neighbor frequency.





Polyadenylic Acid Synthesis

The RNA synthesis data indicated that an additional activity might be present in the RNA polymerase reaction. These indications were: 1) With denatured DNA there was significant incorporation of AMP in the absence of the NTPs. 2) There was a doubling in activity with denatured DNA when Mn2+ replaced Mg 2+, but with native DNA there was little change. 3) There appeared to be a biphasic rate of AMP







61


incorporation, one rate at early times and another at late. These,;TeBits suggested poly(A) might be synthesized early in a2reaction mixture containing denatured DNA.


Assay Requirements

The standard reaction mixture for poly(A) synthesis was the same as that for the RNA polymerase assay (see METHODS) ,except the NTPs were omitted.


DNA
2+
With Mg as cofactor, AMP was incorporated with

denatured DNA in the absence of the NTPs (Table 4). With denatured DNA the reaction was saturated at 0.2 A260/ml (Figure 17), or about half that required to saturate the reaction in the presence of NTPs (Figure 7). The maximum AMP incorporated with native DNA did not exceed 5 percent of the AMP incorporated with denatured DNA.

With Mn2+ as cofactor, there was a 2-fold increase in AMP incorporation with denatured DNA as compared with Mg 2+, but the denatured DNA still saturated the reaction mixture at 0.2 A260/ml (Figure 18). Native DNA supported a much lower rate of incorporation ranging from 8 percent to 15 percent of the activity with denatured DNA.


Substrate

AMP incorporation in the absence of the NTPs was first detected with denatured DNA (Table 4). In the
2+ 2+
presence of Mn rather than Mg only half of the AMP







62


LQ
rd ro
a) -P


0 ro P4 r.


CL4
E-4
Z
mob
0


z U
4
4-)
Q)
r-I ro W 0 -li m r: r-q E-1 0

z
ro
Q)
0 t7) 4
Cy M -H 4J Z rd
0 rd Q)
-P ra
0 4
0
E-1



z x u
0 -ri 4
E-i
0 ro E-4 -r-I -4 H 4-) r-i E-1 0 0 rd U)
z H
Q)
ro >
(d 4-) ro co
111 0 r. r
W 4-) 4 0 H (n (L)
0 Ic:
tM rd 4J


salowd 031VUOddODNI dV4V
ro 9 a) z

Lo rc$ tr) z









63










CN I
In r-i a)
P4 U rO E-i r Z Z 4
44 0
0 r-q
0

z
LO 4
C:k



E-i rl 0
-q U)
z fd
0 -P
rT4 0 >
< W 4-3 ro
a) Q)
4 4-) 0 0 m
4-) 4

z -H 'Z
r4 41 r
a rq -H
E-1 r. Q)
0
rd
z
Z
0
H 4 rd Q)
E-q z r-I
ro rd b) 4 r E-1 (d (1) (d H U) -rq
E-1 (0 4
4 4-3
4C 4J W
z En r ro
(13 r-I r-q 0 0 Z 04 Ef) 10 00 .,A
rz z ro
4 >t
0 (10
z)) U) -P
SGIOLud G3.A.VUO&!O3NI d'14V V-i w M







64


incorporation on denatured DNA was dependent upon the

other NTPs (Table 6). In contrast essentially all the

incorporation of radioactive UMP required the other NTPs.

These results were consistent with the simultaneous synthesis of polyadenylic acid and RNA. Furthermore, they suggested that homopolymer synthesis might be limited to

poly(A).

A titration of the NTP requirement indicated that

at low NTP concentrations (0.025 mM) AMP incorporation was inhibited, whereas at higher NTP concentrations AMP

incorporation was stimulated (Figure 19). Except for the

* activity observed with no NTPs, the stimulatory portion

of this titration resembled that with native DNA (Figure 11).

Addition of GTP, CTP or UTP individually at 3 mM resulted

in 80 percent inhibition of AMP incorporation (Table 7).

--The addition of UTP at 0.3 mM (12% of the standard assay ..concentration) inhibited AMP incorporation to the same degree. The inhibitory effect of low NTP concentrations

was observed upon addition of the NTPs before or after
addition of the enzyme and incubation of the reaction mixture (Table 8). If 0.025 mM NTPs (1% standard NTP concentration) were added just after enzyme addition, after 1 min. incubation or after 5 min. incubation at 301, in all cases it resulted in inhibition of AMP incorporation

(Table 8). The total AMP incorporated per reaction

increased the longer the addition of NTPs was delayed,






65









TABLE 6

AMP AND UMP INCORPORATION BY RNA POLYMERASE




A standard reaction mixture containing 0.37 A260 denatured DNA, 0.4 pmoles MnC12, either 0.10 moles labeled ATP or
0.05 moles labeled UTP and 4 pg enzyme.



Labeled Nucleotide Incorporateda
System (pmoles)
14C-AMP 14C-UMP



Complete 381 22

Complete plus NTPs 913b 458c




aTotal radioactive nucleotide incorporated per reaction
mixture.
bNTPs contained 0.25 moles each: GTP, CTP and UTP. CNTPs contained 0.25 moles each: GTP, CTP and ATP.






66







0 400
0

E
O.O I

0
w 200 T
o T
CL
M 0

0




0 2 5

NTPs mM






Figure 19. NTP TITRATION WITH DENATURED DNA

Assayed in a standard reaction mixture containing
0.037 A260 denatured DNA, 0.5 moles MnCl2 5 pg
260
RNA polymerase and pooled NTPs (GTP, CTP, and UTP)
as indicated (mM each).








67






TABLE 7

INHIBITION OF POLYADENYLIC ACID SYNTHESIS BY NUCLEOTIDES




RNA polymerase activity measured in a standard reaction !mxture containing 0.037 A260 denatured DNA, 0.05 moles 4iCl2, 5 pg enzyme, and nucleotides as indicated.



Additional Nucleotides AMP Incorporateda (pmoles)



GTP, CTP, UTP (0..25 moles each) 619

None 210
UTP (0.3 moles) 40

GTP (0.3 Pmoles) 31

CTP (0.3 moles) 43

UTP (0.03 Pmoles) 50




a Total pmoles AMP incorporated per reaction mixture.







68




TABLE 8

EFFECT OF DELAYED ADDITION OF NTPs ON
POLYADENYLIC ACID SYNTHESIS




A standard reaction mixture containing 0.5 uimoles MnC12,
0.037 A260 denatured DNA, 5 ig enzyme and NTPs as indicated were used. Delayed addition of the NTPs was at times indicated during a standard 20 min. incubation. NTPs contained GTP, CTP and UTP.



NTPs Added AMP Incorporateda
NTPs Added
pmoles %



None 160 40

0.25 moles each, 0 min. 400 100

0.0025 moles each,

0 min. 52 13
1 min. 60 15
5 min. 88 22



aTotal pmoles incorporated per reaction mixture.







69


consistent with the synthesis of poly(A) before NTP addition.


Enzyme

Enzyme was required for AMP incorporation and

increased as a linear function of the enzyme protein present (Figure 20). Replacement of denatured DNA with native DNA caused greater than a 90 percent decrease in AMP incorporation. Therefore, the synthesis of poly(A) was directly proportional to the enzyme level and the enzyme preferred denatured DNA.


'Product Characterization

The acid-insoluble product synthesized with native or denatured DNA with either Mg2+ or Mn2+ as cofactor remained insoluble after heating at 1000 in 1 percent SDS for 5 min. (Table 9). As anticipated, if poly(A) longer than 12 nucleotides was accumulated in the reaction with denatured DNAamore acid-insoluble product was synthesized and it too remained acid-insoluble after treatment.

Nearest neighbor frequency analysis of products synthesized from [a-32p]ATP in the.absence of the NTPs resulted in a 0.90 ApA frequency (Table 10), indicating poly(A) synthesis. Incorporation into CpA, GpA and UpA accounted for 10 percent of the label incorporated.

The average chain length of the product was estimated by the ratio of AMP to adenosine after alkaline hydrolysis. The chromatographed AMP fraction contained 2,200 cpm,






70







0400
4OO
0
E


C w
1

0 200

00


00
z


oa...... ,'


0 3 6
PROTEIN pg




Figure 20. POLYADENYLIC ACID SYNTHESIS AS A
FUNCTION OF ENZYME CONCENTRATION

Assayed in a standard reaction mixture containing
0.5 mole MnCl 0.37 A 60 DNA, either native (solid circles or dena ured (solid triangles), and enzyme protein as indicated.







71








TABLE 9

EFFECT OF HEATING AND SDS ON
RNA POLYMERASE PRODUCT




Standard reaction mixture with 0.37 A260 DNA, and either
1.0 moles MgCl2 or 0.4 moles MnCI2 as indicated. Heating was for 5 min. at 1000. SDS was 1% where indicated. Each assay contained 6.5 jig enzyme.



Components in Reaction Mixture AMP Incorporated poleses*
Template Metal Control Heated Heated in SDS


Native Mg 660 610 625

Native Mn 627 632 630

Denatured Mg 805 800 840

Denatured Mn 1160 1160 1190




Total radioactive nucleotide incorporated per reaction mixture.







72







TABLE 10

NEAREST NEIGHBOR FREQUENCY ANALYSIS OF
POLYADENYLIC ACID PRODUCT




A standard reaction mixture containing 0.37 A260 denatured DNA, 0.10 pmoles [-32P]ATP (specific activity 30 mCi/mmole),
0.4 moles MnC12 and 10 yg enzyme was incubated at 300 for 60 min. 1% SDS was added to the reaction, it was passed over sephadex G25, material in the excluded volume was hydrolyzed in 0.3 M KOH for 18 h at 370, adsorbed to activated charcoal and the eluted nucleotides lyophilized, resuspended in H20 and resolved by paper electrophoresis (see METHODS).



CPM
CpA ApA GpA UpA
Added Recovereda



11,000 4,500 0.01 0.90 0.04 0.05



aSome of the sample was lost during lyophilization.







73










TABLE 11

AVERAGE CHAIN LENGTH OF POLYADENYLIC ACID




A standard reaction mixture was used containing 0.037 A260 denatured DNA, 0.5 moles MnCI2, no NTPs and 12 jg RNA polymerase. The reaction was incubated for 60 min. at 300. Yeast RNA (1 mg) was added and the product precipitated by the addition of 0.3 ml of cold 1.0 M HCl04 containing 5 mM sodium pyrophosphate. The ppt. was washed 4X with 1 ml of cold 0.5 M HCI04 and then alkaline hydrolyzed and electrophoresised (see METHODS).




Compound CPM



AMP 2,200

Adenosine <22







74

whereas the radioactivity detected in the adenosine fraction did not exceed background (Table 11). The sensitivity of the assay for adenosine was 22 cpm; therefore the lower estimate of the average chain length for poly(A) was 2,200/ 22, or greater than 100 nucleotides per chain after 60 min. at 300.


Summary

Maize RNA polymerase synthesized polyadenylic acid in the presence of denatured DNA, ATP and metal, preferably Mn 2+. There was greater than a 17-fold preference for ATP over UTP as substrate for homopolymer synthesis. Low levels of the NTPs as a mixture or added individually inhibited poly(A) synthesis, independent of the time of addition. Poly(A) synthesis was directly proportional to the amount of enzyme protein added. The product remained acid-insoluble after boiling with.SDS. The average chain length of the product accumulated after 60 min. incubation was greater than 100 nucleotides and the ApA frequency was

0.90.





Comparison of RNA and Polyadenylic Acid Synthesis


The two AMP incorporating activities measuring RNA

and poly(A) synthesis were apparently catalyzed by the same enzyme; RNA polymerase II (69). The evidence for this






75


conclusion was indirect, since the enzyme preparation was not homogeneous. The data presented thus far in support of one enzyme catalyzing both syntheses includes: 1) Both activities were eluted simultaneously from DEAE-cellulose by an (NH4)2So4 gradient with a constant RNA and poly(A) activity ratio in the active fractions. 2) Both activities required DNA for AMP incorporation. 3) Both activities had the
2+ 2+
same Mg and Mn cofactor optima. 4) The K for ATP was
m
the same with native and denatured DNA. Additional comparisons of RNA and poly(A) synthesis were made to further associate the accumulation of both products with one enzyme. These included inhibitor activities, nuclease sensitivity of products as a function of time and template specificity.


Effect of Inhibitors

Three types of inhibitors of RNA polymerase were

investigated, those that bind specifically to the enzyme protein such as a-amanitin and rifampicin; those that bind to the template such as actinomycin D; and those that compete with the substrate such as cordycepin. The inhibitors were tested both in the presence and absence of the NTPs, therefore measuring the sensitivity of both RNA and poly(A) syntheses.

Low concentrations of a-amanitin inhibited both RNA and poly(A) activity equally (Table 12). A titration of RNA polymerase activity with increasing levels of a-amanitin







76




TABLE 12

INHIBITOR SENSITIVITY OF MAIZE RNA POLYMERASE




A standard reaction mixture contained 0.037 A260 denatured DNA, 0.5 moles MnCl2, 0.05 moles ATP, 5 pg enzyme and inhibitors as indicated added at zero times.




Inhibitor Concentration Percent of Control
Plus NTPsa Less NTPsb


a-amanitin 0.1 PM 6 7
1.0 uM 1 2

Actinomycin D 7.0 PM 14 128
70.0 PM 1 87

Cordycepin 0.13 mM 100 101
0.26 mM 100

Rifampicin 50.0 ug/ml 100 110




a336 pmoles AP incorporated equaled 100%. b138 pmoles AMP incorporated equaled 100%.







77


indicated 50 percent inhibition at 1 x 10- M (Figure 21). Since a-amanitin is specific for the type II eukaryotic RNA polymerase (32), this inhibition of activity indicated that the maize enzyme was a type II RNA polymerase and that it catalyzed both RNA and poly(A) synthesis (69).

Actinomycin D inhibited AMP incorporation in the

presence of the NTPs (Table 12). However, in the absence of the NTPs, AMP incorporation was stimulated. Even at very high actinomycin D concentrations (70 iM), there was only a slight inhibition of poly(A) synthesis. Therefore, the sensitivity of the RNA polymerase to actinomycin D was dependent upon the presence of the NTPs. In the presence of the NTPs, AMP incorporation was inhibited 50 percent at 2 pM actinomycin D and was inhibited 95 percent at 50 vM (Figure 22).

The inhibition by a-amanitin and the resistance to cordycepin and rifampicin excluded the presence of two potential enzyme contaminants (Table 12). The maize NTP: exotransferase is resistant to a-amanitin and sensitive to cordycepin while bacterial RNA polymerases are resistant to .a-amanitin and sensitive to rifampicin. Therefore the presence of NTP: exotransferase activity from maize tissue and bacterial RNA polymerase activity from contaminating bacteria were ruled out.

Utilizing the resistance of purine-purine

phosphodiester linkages to pancreatic ribonuclease (54),






78






100








e
-50




0
W U CL




0 5 10 20
ALPHA AMANITIN XIO M

Figure 21. ALPHA-AMANITIN TITRATION OF RNA POLYMERASE Assayed in a standard reaction mixture containing 1.0 mole MgC1 2, 0.37 A260 native DNA, 18 ug RNA polymerase and a-amanitin as indicated. Inhibitor was added just prior to addition of enzyme to the reaction mixture.







79







10oo0,












50
U





W

00





1 I2 34 50 100

ACTINOMYCIN D pM


Figure 22. ACTINOMYCIN D TITRATION OF RNA POLYMERASE Assayed in a standard reaction mixture containing 1.0 mole MgC12, 0.37 A 260 native DNA, 18 ig RNA polymerase and actinomycin D as indicated. Inhibitor was added just prior to addition of enzyme to the reaction mixture.







80

we determined the amount of poly(A) accumulated with denatured DNA in a standard reaction mixture as a function of Stime. 'MP incorporation in the absence of the NTPs was not a-ffected by the ribonuclease treatment (Figure 23, panel B). In data not shown here AMP incorporation in a standard reaction mixture containing denatured DNA and

4 pg ribonuclease showed no inhibition of incorporation, consistent with the exclusive synthesis of poly(A). In the presence of the NTPs, there was a decrease in acidinsoluble AMP when the product was treated with ribonuclease (Figure 23, panel A). However, the 10-min. product was ;-znly 10 percent sensitive to treatment whereas the 60-min. product was 45 percent sensitive. This result suggests early synthesis of poly(A) and later synthesis of RNA. Template Requirement

Both calf thymus and maize DNA supported RNA and

poly(A) synthesis (Table 13). In the absence of the NTPs, where only poly(A) was synthesized, denatured maize and calf thymus DNA supported equal AMP incorporation. Since poly(A) synthesis was supported by DNA from both species, the synthesis of poly(A) was not an artifact of the heterologous system (calf thymus DNA and maize polymerase). The lower AMP incorporation in the complete system observed on denatured maize DNA as compared with denatured calf DNA reflects the presence of native DNA in the heated maize preparation; note the low hyperchromicity of the maize DNA.








0 81


Q4

0 0 -P w
u rd
IPA 4J (d
LO 0 -P
ul 0 4 M
p CD Ic: 0 Q)
-P H 4 rd 4 4J
C to (D
04 4 M
P4 -P (
Z Z
E-i C) a) of Z z M C)
pq rd ro r-A
Z 4 Q) r.
N : -,I
CIO P4
rzj 0
0 Q) m Q) 04 -P E-4 ro rd pq
F: -P
C C=z rq
ID4 0)


r
M Ln 24 0
0 z rd p rd

4-) T)
rZ4 -r-i "I j 0 Q) r
r-l P4 0 m p ro IQ E-f 4-) Z r. -rq
> 0 4-3
U : T5 U E-q 0 a) -r-i
F-i Q) 4 > 4-) m p -W 0 r
z : -li F (1)
w 41


to r-A 4 Q4
0
4-) )4 Ul u Q) CN
Z 4
0 0 Q)
PQ 4 P4 4-) r-q
H E-1 X 4 U
P4 rC; Z -H -P 4

rd 4-) r. CY) Z -4 0 rO Z C14 m : -rq (1) (1) 4-) 4J 4-3 P4
(1) En 4 U m 0
0 4 (d Q
0 J 0 >t
OD Q Q
rk, (a rd -H ro
0.4 Q) a) StIlOUXI (3-3--UV80d.803lNAI I dvL' lug V rd 4J 4 -P
a) .- CO 0 M >1 r4u 0
rd r--f -1-1 rd -H w u rd -H ro 9 0 r
-rA rd -H






82









TABLE 13

COMPARISON OF CALF THYMUS AND MAIZE DNA AS
TEMPLATES FOR RNA POLYMERASE




Assayed in a standard reaction mixture containing 0.5 moles MnC12, 0.037 A260 DNA and 6.4 yg RNA polymerase


DNA AMP Incorporateda (pmoles)
DNA ol
Complete Less NTPs


Native Calf Thymus 219 29

Native Maize 260 46


Denatured Calf Thymusb 400 160

Denatured Maizec 300 163




aTotal pmoles incorporated per reaction mixture. bHyperchromicity 26%.
Hyperchromicity 1126%.
CHynerchromicity 11%.






83


If RNA polymerase catalyzed two reactions, RNA

synthesis and poly(A) synthesis, and if both reactions required a template, then no AMP incorporation on poly(dAT), the alternating copolymer, would be expected in the absence of UTP. As seen in Table 14, AMP incorporation on poly(dAT) was observed only in the presence of UTP.

Poly(dAdT), the complementary homopolymers, were

also templates for AMP and UMP incorporation. AMP incorporation was stimulated 2-fold by the addition of UTP, suggesting synthesis of poly(U) on a poly(dA) template. However, when UMP incorporation was measured directly (with 14C-UTP) little product accumulated unless ATP was added to the reaction mixture, suggesting that poly(A) synthesis was required for UMP incorporation. Incorporation with the complementary homopolymers was sensitive to a-amanitin indicating that the RNA polymerase was catalyzing the ATP stimulated UMP incorporation.


Summary

RNA and poly(A) synthesis by RNA polymerase II was

confirmed by the sensitivity of both to a-amanitin. Ribonuclease resistance suggested that the early product was poly(A). Requirements for poly(A) and RNA synthesis with several deoxyoligomers demonstrated that template was required for both poly(A) and RNA synthesis. The differential 'sensitivity to actinomycin D indicated that







84




TABLE 14

RNA POLYMERASE ACTIVITY WITH SYNTHETIC TEMPLATES




Enzyme activity assayed in a standard reaction mixture containing 0.5 moles MgCl2, 0.037 A260 DNA, either 0.1 mole labeled ATP or 0.05 mole labeled UTP and 4 ug RNA polymera s e.



Nucleic Acid Substrate Incorporation (pmole
AMP UMP


Poly(dAT) 14C-ATP 13

Poly(dAT) C-ATP + UTP 1,090

Poly(dAdT) 14C-ATP 104

Poly(dAdT) 14C-ATP + UTP 211

Poly(dAdT) 14C-UTP 4

Poly(dAdT) 14C-UTP + ATP 40

Poly(dAdT) 14C-UTP + ATP (a-aman)b 0.2




aTotal radioactive nucleotide incorporated per reaction mixture.

ba anitin added to 20 g/ml.
a-amanitin added to 20 uag/ml.







85


the polymerase utilized different sequences in the DNA but not necessarily different DNA molecules for RNA and poly(A) synthesis.














DISCUSSION


Enzyme Preparation

Studies of both product characterization and the

initiation of transcription required an enzyme of a much higher specific activity than the soluble maize RNA polymerase purified by Stout and Mans (27). In addition, the net yield of active polymerase had to be increased and the purification procedure made more convenient and rapid for .1 effective experimental progress. These goals were achieved. The RNA polymerase specific activity was increased 10-fold, the net yield was more -than doubled, and the time required for each preparation was reduced from 7 days to 10 hours. These improvements resulted from 4 major alterations in the procedure of Stout and Mans (27), including changes in:

1. the storage of the starting material; 2. the homogenization procedure; 3. the salt equilibration of the soluble proteins; 4. the DEAE-cellulose chromotography procedure.

Previously (27), adequate grain was germinated under

running water andafter 5 days, the seedlings were harvested just before each enzyme preparation. In contrast, after large scale germination and harvest (1 to 3 kg) followed by storage of the seedlings at -761 (Revco freezer) in 86







87


aluminum packets, each enzyme preparation required only the removal of a weighed packet of tissue from the freezer just before homogenization. The specific activities of the homogenates from freshly harvested tissue and from tissue stored at -760 were identical.

In the original procedure (27), the shoots and roots were pulverized under liquid nitrogen and then, in small batches, passed through a French pressure cell. This was replaced by rapid homogenization, in one batch, in a Waring blender. During homogenization, the enzyme was protected from oxidation (foaming) with 50 mM 2-mercaptoetLhanol. The enzyme specific activity of the blender treated material (Table 1) was identical to material from the French pressure cell (27).

The removal of (NH4)2S04 from the salt-precipitated enzyme fraction by a 4 h dialysis against Buffer R (27), was replaced by a 20 min. gel filtration procedure. The Sephadex G50 was equilibrated and the proteins eluted in

-the excluded volume with 0.2 M (NH4)2 so4' At this salt concentration all RNA polymerase was bound to the DEAEcellulose column, but only a small fraction of the total protein was bound (illustrated by A280 in Figure 3).

Previously (27), RNA polymerase was eluted with a

shallow Tris-HCl gradient (500 ml, 0.05 to 1.0 M). This was replaced by a steep (NH4)2 so4 gradient (60 ml, 0.2 to 1.0 M), that eluted concentrated, highly active, RNA polymerase




Full Text
32
TABLE 1
STEPS IN RNA POLYMERASE PURIFICATION
A standard reaction mixture contained 0.37 A_,n DNA, 1.0 ymole
Zb U
MgC2/ and enzyme ,as follows: 96 yg 200,000 x g supernatant,
500 yg 50% ASP, 250 yg sephadex G50, or 3 to 5 yg DEAE-
cellulose fraction.
Step
Protein
Specific
Activity
Total
Activity
Yield
(mg)
(nmoles AMP/mg)(units)
(%)
200,000 x g Supernatant2
507+100
0.570.12
29090
100
50% ASP2
207 50
0.72+0.3
19560
6730
Sephadex G5Qa
250 50
0.700.07
17520
6010
DEAE-cellulo s e
Q
Gradient elution
2.0
66.0
132
55
ci
Batch elution
5.0
40.0
200
69
*
3.
Average of 3 preparations.
^Flowthrough contained less than
5% of added
RNA polymerase
activity.
r ...
'"'Gradient elution, peak tube specific activity, 10:4 nmoles
AMP/mg; protein, 0.2 mg/ml.
^Batch elution (0.4 M (NH
:,)oSO),
peak tube specif ic activity,
90 nmoles AMP/mg protein 0.5 mg/ml.


AMP INCORPORATED pmoles
70
PROTEIN pg
Figure 20. POLYADENYLIC ACID SYNTHESIS AS A
FUNCTION OF ENZYME CONCENTRATION
Assayed in a standard reaction mixture containing
0.5 ymole MnCl-/ 0.37 DNA, either native
(solid circlesf or denatured (solid triangles),
and enzyme protein as indicated.


Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
PURIFICATION OF A EUKARYOTIC RNA POLYMERASE II
THAT SYNTHESIZES POLYADENYLIC ACID
By
Robert Henry Benson
December, IS72
Chairman: Rusty J. Mans, Ph.D.
Major Department: Immunology and Medical Microbiology
The in vitro catalytic activities of DNA-dependent
RNA polymerase II, isolated and purified from rapidly
growing 5-day-old maize seedlings (WF9 x Bear 38, waxy),
have been examined. An enzyme purification procedure is
described utilizing homogenization, differential centri
fugation, salt precipitation and ion exchange chromoto-
graphy that resulted in 1,000-fold purification with
70 percent recovery in 6 to 10 hours. The enzyme had a
specific activity greater than 100 nmoles AMP/mg/20 min.
at 30, and contained one major band and several minor
bands on polyacrylamide gel electrophoresis. Incorpora
tion of AMP into acid-insoluble material required DNA,
4 nucleosidetriphosphates: GTP, CTP, UTP, ATP (NTPs),
2+ 2+
and metal, either Mn or Mg Alkaline hydrolysis of
xi


6 6
NTPs mM
Figure 19. NTP TITRATION WITH DENATUREDDNA
Assayed in a standard reaction mixture containing
0.037 denatured DNA, 0.5 timles MnCl- 5 yg
RNA polymerase and .pooled NTPs (GTP, CTP, and UTP)
as indicated (mM each).


27
Protein Determination
Protein was measured on samples precipitated with
10 percent trichloroacetic acid by the method of Lowry
et al. (62), using BSA as a standard.
Ribonuclease Treatment
Pancreatic ribonuclease (80 yg/ml), heated at 80
for 10 min. to inactivate DNase, was added to 0.1 ml aliquots
of an incubated reaction mixture to a final concentration
of 20 yg/ml, and incubated for an additional 10 min. at
30 (54). Acid-insoluble material remaining was determined
on filter paper disks with the polymerase assay wash
procedure (see polymerase assay).
Alkaline Hydrolysis
Product, isolated by sephadex gel filtration or by
KC10^ precipitation from an incubated reaction mixture,
was suspended in 1.0 ml of 0.3 M KOH and incubated at 37
for 18 h in a stoppered test tube. After the 18 h hydrolysis,
the solution was acidified with 7 0 percent HC10^ and the
precipitate removed by centrifugation. The supernatant
was neutralized with 0.3 M KOH and the salt was removed
from the hydrolysate by charcoal adsorption (63). The
charcoal column was washed successively with water ( 3 ml)
and 0.1 M NH^OH (5 ml) and the nucleotides eluted with
5 ml of ethanol-NH^OH (ethanol-conc NH^OH-^O, 2:1:2 by
volume). The eluted fraction was lypholyzed to dryness and
resuspended in water.


44
TABLE 4
ASSAY REQUIREMENTS FOR MAIZE RNA POLYMERASE
A standard reaction mixture contained 0.37 A,n DNA, 1.0 ymole
A DU
MgCi^ or 0.5 ymoles MnCl^ and 5 yg RNA polymerase.
System
AMP Incorporated3 (pmoles)
Native DNA Denatured DNA
Complete 235 327
Complete less DNA 0 1
2+
Complete less Mg 0 1
Complete less Mg^+, plus Mn^+ 300 551
Complete less NTPs 10 58
Complete less enzyme 0 0
lTotal radioactive AMP incorporated per reaction mixture.


30
0.10 M Tris-HCl, pH 8.0, 1.0 mM MgC^/ and 50 mM 2-mercap-
toethanol; Buffer E, 0.05 M Tris-HCl, pH 7.6, 10 mM
2-mercaptoethanol and 0.20 M (NH^^SO^; Buffer R, 0.05 M
Tris-HCl, pH 7.6, 10 mM 2-mercaptoethanol. Buffers were
made immediately before use, using warm glass~distilled
water to minimize gas bubbles in the column eluates passing
through the spectrophotometer flowcell.
Saturated ammonium sulfate was kept at 4 with
crystals visible in the bottom of the bottle. Before use
in protein precipitations, concentrated NH^OH was added
drop-wise until a 1:20 dilution of the (NH^^SO^ was pH 8
at 25 (67). Sodium chloride-sodium citrate buffer (SSC)
1 X, was 0.15 M NaCl, 0.015 M sodium citrate, pH 7.3 at
25 (68) .


to
Assayed in a standard reaction mixture containing 1.0 ymole MgCl, 0.37 Anative DNA and
4 yg RNA polymerase. The 14C-ATP specific activity was as follows: panel A; 6.5 Ci/mole,
panel B; 4.5 Ci/mole, panel C; 2.1 Ci/mole. The ATP concentrations were as follows: 1.0 mM
(solid circles), 0.5 mM (open circles) and 0.25 mM (solid triangles).
108


38
c)
Figure 6. POLYACRYLAMIDE GEL ELECTROPHORESIS OF
DENATURED RNA POLYMERASE
Samples were denatured in 0.1% SDS, 0.14 M 2-mercapto-
ethanol at 37 and run on 10% polyacrylamide gels
containing 0.1% SDS at pH 7.0 (see METHODS). Gel A
contained 20 yg peak RNA polymerase (eluted at 235 ml,
Figure 4). Gel B contained 6.2 yg denatured BSA.
Gel C contained denaturing buffer only.


AMP INCORPORATED pmoles
Figure 8. DNA TITRATIONS WITH MANGANESE AS COFACTOR
Assayed in a standard reaction mixture containing 0.5 pmoles
MnCl2/ 4 pg RNA polymerase and DNA as indicated; native (solid
circles) or denatured (solid triangles).


59
TABLE 5
NEAREST NEIGHBOR FREQUENCY ANALYSIS OF PRODUCT
SYNTHESIZED WITH DENATURED DNA
A standard reaction mixture containing 0.37 denatured
DNA, 0.10 nmoles [a-32p]ATP (specific activity 30 mCi/mmole) ,
0.4 ymoles MnC^ and 10 ng enzyme was incubated at 30 for
60 min. 1% SDS was added to the reaction, it was passed
over sephadex G25, the material in the excluded volume was
hydrolyzed in 0.3 M KOH for 17 h at 37, adsorbed to acti
vated charcoal and the eluted nucleotides lyophilized, re
suspended in H2O and resolved by paper electrophoresis.
(see METHODS).
CPM
Added Recovered9,
CpA ApA GpA UpA
55,000 27,600
0.25 0.44 0.18 0.13
aSome of the sample was lost during lyophilization.


90
rat and calf thymus enzymes (32) was not observed for
maize RNA polymerase (Table 4). Rather than the 5-to
14-fold preference expected, the maize enzyme exhibited,
2+
at most, a 2-fold preference for Mn This low ratio
of Mn-dependent to Mg-dependent activity could be a unique
property of maize RNA polymerase, or it could reflect a
variable rate of polymerase degeneration. If the Mg-
dependent activity was more labile than the Mn-dependent
activity, then the low Mn/Mg activity ratio may have
resulted from the rapid purification procedure which per
mitted the maize Mg-dependent activity to remain active.
All the type II RNA polymerases have the unique property
of a-amanitin sensitivity (32). The evidence involving
enzyme purification and assay characteristics suggested,
and the inhibition by a-amanitin (Figure 21) confirmed
the inference, that the maize enzyme was a type II poly
merase (45). More importantly, a-amanitin inhibition
indicated poly(A) synthesis was also catalyzed by the
RNA polymerase II (Table 12). The synthesis of both RNA
and poly(A) by the same enzyme was supported by the co
purification of both activities during DAE-cellulose
chromotography, and the identical metal optima and the
identical K for ATP. Therefore, the same enzyme was
m
responsible for both activities.


GRADIENT
SOLUTIONS
}>
o
rn
m
c
>
o
H
o
PI
o
r
C
o
r?
A39
<
2
n
l
VIALS
ICE
BUCKET
V
TO
LIQUID
NITROGEN
Figure 2. DEAE-CELLULOSE GRADIENT ELUTION ASSEMBLE
to
<_n
4


107
tlie amount of cold ATP added. The net effect is an
increase in the specific activity of the labeled ATP.
However, since the ATP concentration is no longer satu
rating the reaction, the total pmoles of AMP incorporated
decreases. This decrease in incorporation with lower ATP
concentrations was a consistent percentage for all time
points examined out to 40 min. (Figure 25). If the ATP
concentration was decreased from 1.0 mM to 0.5 mM, 86 per
cent of the maximum rate remained; if the ATP concentration
was decreased to 0.25 mM, 68 percent of the maximum rate
remained. Since the labeled ATP was held constant as the
total ATP concentration was decreased there was a 2-fold
increase in specific activity with 0.5 mM ATP, and a 4-fold
increase with 0.25 mM. The. combined result of the decreased
amount of AMP incorporated and the increased labeling of
that which was incorporated was a 72 percent increase in
cpm for 0.5 mM ATP and a 164 percent increase in cpm for
0.25 mM ATP. Therefore, the labeled substrate was conserved
and more cpm were incorporated into the RNA polymerase
product.


J200r
O
E
Ol
DMA A26o/ml
Figure 18. DNA TITRATIONS WITH MANGANESE IN THE ABSENCE OF NTPs
Assayed in a standard reaction mixture containing 0.5 ymole MnCl^
4 yg RNA polymerase and DNA, either native (solid circles) or de
natured (solid triangles), as indicated.
cn
co


28
Paper Electrophoresis
Samples to be electrophoresised were adsorbed to
paper strips (4 x 30 cm, Whatman #1) by repeatedly apply
ing and drying on one spot (5 to 10 mm dia). The paper
strips were then subjected to electrophoresis in 0.025 M
sodium citrate, pH 3.5, for 4 h at 300 v, 6-8 amps, at
25 in a Universal Electrophoresis. Cell (Buchler Instru
ments) .
Polyacrylamide Gel Electrophoresis
Polyacrylamide gels for native proteins were prepared
and run according to the method of Davis (64) using a
stacking gel and 5.2 percent polyacrylamide gels. The
gels were run at pH 8.8 and stained with aniline blue
black. The 10 percent polyacrylamide gels and denatured
enzyme protein were prepared and run according to the
method of Weber and Osborn (65). The 10 percent gels were
run at pH 7.0 in 0.1 percent SDS and stained with coomassie
brilliant blue.
Materials
DEAE-cellulose, Bio-RAD, Cellex-D (6.1 meq per g,
dry weight), was washed by the method of Peterson and Sober
(66), equilibrated in 0.05 M Tris-HCl, pH 7.6 at 25, and
stored at 4o. Sephadex G50, medium grade, Pharmacia, was


64
incorporation on denatured DNA was dependent upon the
other NTPs (Table 6). In contrast essentially all the
incorporation of radioactive UMP required the other NTPs.
These results were consistent with the simultaneous syn
thesis of polyadenylic acid and RNA. Furthermore, they
suggested that homopolymer synthesis might be limited to
poly(A).
A titration of the NTP requirement indicated that
at low NTP concentrations (0.025 mM) AMP incorporation
was inhibited, whereas at higher NTP concentrations AMP
incorporation was stimulated (Figure 19). Except for the
activity observed with no NTPs, the stimulatory portion
of this titration resembled that with native DNA (Figure 11)
Addition of GTP, CTP or UTP individually at 3 mM resulted
in 80 percent inhibition of AMP incorporation (Table 7).
The addition of UTP at 0.3 mM (12% of the standard assay
concentration) inhibited AMP incorporation to the same
degree. The inhibitory effect of low NTP concentrations
was observed upon addition of the NTPs before or after
addition of the enzyme and incubation of the reaction mix
ture (Table 8). If 0.025 mM NTPs (1% standard NTP con
centration) were added just after enzyme addition, after
1 min. incubation or after 5 min. incubation at 30, in
all cases it resulted in inhibition of AMP incorporation
(Table 8). The total AMP incorporated per reaction
increased the longer the addition of NTPs was delayed,


83
If RNA polymerase catalyzed two reactions, RNA
synthesis and poly(A) synthesis, and if both reactions
required a template, then no AMP incorporation on
poly(dAT), the alternating copolymer, would be expected
in the absence of UTP. As seen in Table 14, AMP incorpo
ration on poly(dAT) was observed only in the presence of UTP,
Poly(dAdT), the complementary homopolymers, were
also templates' for AMP and UMP incorporation. AMP incor
poration was stimulated 2-fold by the addition of UTP,
suggesting synthesis of poly(U) on a poly(dA) template.
However, when UMP incorporation was measured directly
14
(with C-UTP) little product accumulated unless ATP was
added to the reaction mixture, suggesting that poly(A)
synthesis was required for UMP incorporation. Incorporation
with the complementary homopolymers was sensitive to
a-amanitin indicating that the RNA polymerase was catalyzing
the ATP stimulated UMP incorporation.
Summary
RNA and poly(A) synthesis by RNA polymerase II was
confirmed by the sensitivity of both to a-amanitin. Ribo-
nuclease resistance suggested that the early product was
poly(A). Requirements for poly(A) and RNA synthesis with
several deoxyoligomers demonstrated that template was
required for both poly(A) and RNA synthesis. The
differential'sensitivity to actinomycin D indicated that


AMP INCORPORATED pmoles
49
Figure 9. MAGNESIUM TITRATION WITH
NATIVE AND DENATURED DNA
Assayed in a standard reaction mixture
containing 0.37 A?60 DNA; native (solid
circles) or denatured (solid triangles),
6.4 yg RNA polymerase and MgC^ as
indicated.


78
Figure 21. ALPHA-AMANITIN TITRATION OF RNA POLYMERASE
Assayed in a standard reaction mixture containing 1.0 jimole
MgC^r 0.37 &260 nat^-ve 18 yg RNA polymerase and
a-amanitin as indicated. Inhibitor was added just prior to
addition of enzyme to the reaction mixture.


95
Poly(A) Synthesis and the Initiation
of Transcription
A major unsolved problem in eukaryotic transcription
is the initiation of RNA synthesis, including the recogni
tion of the binding site on DNA and selection of the
proper strand for transcription.. The discovery of poly (A)
synthesis by maize RNA polymerase II indicated the
phenomenon of reiterative poly(A) synthesis by prokaryotes
(E. cold.) could be extended to eukaryotes. Unlike the
prokaryotes, maize RNA polymerase II synthesizes poly(A)
and RNA during the same reaction. In prokaryotes, ATP
and GTP are found at the 5' terminus of newly synthesized
RNA. GTP initiation maybe an artifact of the in vitro
system (71), suggesting ATP is the first base incorporated
during initiation of transcription. Perhaps it is the
beginning of a short poly(A) strand, or perhaps the remnant
of a longer discontinuous poly(A) strand.
Transcription of native DNA is asymmetric (72), that
is, only one strand of the DNA at any one region of the
DNA is transcribed into RNA. If the RNA polymerase had
a binding affinity for only poly(dT)-rich regions on one
strand of the double helix, the complementary strand con
taining poly (dA) would not be bound. Therefore, not only
selection of the proper site, but selection of the proper
strand of DNA, might be attributed to poly(A) synthesis.


conclusion, I would like to thank John Henry Colson and
Bernardine, for keeping me company during all my nights
in the laboratory repeating experiments and typing endless
drafts of this dissertation.
iv


This dissertation is dedicated to my parents,
MaryAnn L. and Henry E. Benson, for their end
less encouragement during my 25 years of
education.


AMP INCORPORATED pmotes
Figure 15. RNA POLYMERASE ACTIVITY AS A
FUNCTION OF ENZYME CONCENTRATION
Assayed in a standard reaction mixture con
taining 0.5 pinoles MnC^? 0.37 A^gg DNA,
either native (solid circles) or"' denatured
(solid triangles), and enzyme protein as
indicated.
~5 *0


61
incorporation, one rate at early times and another at late.
TheserreBuis'suggested poly(A) might be synthesized early
in a ire-actioxi mixture containing denatured DNA.
Assay Requirements
The standard reaction mixture for poly(A) synthesis
was the same as that for the RNA polymerase assay (see
METHODS) except the NTPs were omitted.
DNA
2+
With Mg as cofactor, AMP was incorporated with
denatured DNA in the absence of the NTPs (Table 4). With
denatured DNA the reaction was saturated at 0.2 A2gQ/ml
(Figure 17), or about half that required to saturate the
reaction in the presence of NTPs (Figure 7). The maximum
AMP incorporated with native DNA did not exceed 5 percent
of the AMP incorporated with denatured DNA.
2+
With Mn as cofactor, there was a 2-fold increase
in AMP incorporation with denatured DNA as compared with
2+
Mg but the denatured DNA still saturated the reaction
mixture at 0.2 (Figure 18). Native DNA supported
a much lower rate of incorporation ranging from 8 percent
to 15 percent of the activity with denatured DNA.
Substrate
AMP incorporation in the absence of the NTPs was
first detected with denatured DNA (Table 4). In the
2+ 2+
presence of Mn rather than Mg only half of the AMP


2) <4-14 p
health
center
library.


2
requirements and product characteristics. Following
this initial evaluation, transcriptional control functions,
such as template specificity or the presence or absence
of initiation and termination factors, can be determined.
The DNA-dependent RNA polymerases all require a
DNA template, the four nucleoside triphosphates: ATP,
UTP, GTP and CTP, and metal during the synthesis of RNA
and concomitant liberation of pyrophosphate. The RNA
polymer is synthesized in the 5' to 3' direction. The
new nucleotide is linked via a phosphodiester linkage to
the 3' hydroxyl of the growing RNA chain. As the RNA is
synthesized in the 5' to 3' direction, the template DNA
is transcribed in the 3' to 5' direction. RNA polymerase
activity can be evaluated separately in four operational
steps: binding, initiation, elongation and termination.
Step one is the binding of the RNA polymerase to the DNA
template. Step two is the initiation of an RNA chain
via synthesis of the first phosphodiester linkage. Step
three is the elongation of the new RNA chain. Step four
is the termination of RNA synthesis. Release of the RNA
chain may occur immediately or it may be delayed.
Experimental evaluation of the initial steps,
binding to the DNA template and initiation of RNA syn
thesis, requires an enzyme of high specific activity.
This high activity is required to experimentally detect


AMP INCORPORATED pmoles
Figure 17. DNA TITRATIONS WITH MAGNESIUM IN THE ABSENCE OF NTPs
Assayed in a standard reaction mixture containing 1.0 ymole MgCl2, 4yg RNA polymerase
and DNA, either native (solid circles) or denatured (solid triangles), as indicated.
fO


13
Most studies of organelle transcription therefore utilized
in vivo ..labeling and experimentation.
Structure
RNA polymerase II has been purified from calf thymus,
rat liver, sea urchin, yeast, mouse embryo cells, Hela
cells, xenopus and maize (1,5,27). The subunit structure
of the type II RNA polymerase from calf thymus was evalu
ated on SDS-polyacrylamide gel electrophoresis (38) These
subunits had molecular weights of 215,000, 185,000 and
150,000 and were designated Bl, B2, and B3. Gel densitom
etry indicated a ratio of 1:1:2 between the gel bands
Bl:B2:B3 (39). These subunits apparently came from two
subsets of RNA polymerase II. The first subtype gave
bands Bl and B3, the second subtype gave bands B2 and B3.
There were also 3 smaller subunits of molecular weights
20,000, 30,000 and 40,000. The large subunits Bl, B2 and
B3 appeared to be similar to the 0 and 3' subunits of the
E. coli RNA polymerase. It has been suggested that cr-amanitin
inhibits the type II RNA polymerase by binding to subunit
B3.
Maize RNA Polymerase
Maize RNA polymerase was one of the first eukaryotic
RNA polymerases solubilized (27). Other eukaryotic RNA
polymerases were solubilized from animal tissues (1);
however, other plant RNA polymerases were studied primarily


42
TABLE 3
SALT PRECIPITATION OF RNA POLYMERASE
Each sample contained 0.75 mg protein before 80% saturation
with solid, powdered (NH^^SO^. Just before addition of
salt, DNA was added as indicated, then salt was slowly add
ed while the mixture was stirred on ice for 15 min. The
precipitate was collected by centrifugation (10,000 x g for
10 min.), resuspended and desalted in Buffer R on sephadex
G25. Samples were assayed in a standard reaction mixture
with 1.0 ymole MgCl2 and 0.37 A2gQ DNA.
DNA Added (yg/ml)
% Activity Recovered3
None
13
2.4
20
6.0
30
12.0
38
30.0
46
300.0b
10
a
Control incorporated 185 pmoles AMP.
Very little precipitate obtained.
I


AMP INCORPORATED pmoles
50
Figure 10. MANGANESE TITRATION WITH
NATIVE AND DENATURED DNA
Assayed in a standard reaction mixture
containing 0.37 &260 DNA? native (solid
circles) or denatured (solid triangles),
6.4 \ig RNA polymerase and MnC^ as indicated.


METHODS AND MATERIALS
Methods
RNA Polymerase Purification
The purification of maize RNA polymerase has evolved
through enumerable modifications of that published initially
(27). Each step in the procedure utilized here is indicated
in Figure 1.
Step 1. Preparation of material
Grain (4-10 liters), in a plastic garbage can with a
perforated bottom, was imbibed and germinated under run
ning water (23) for five days until the shoots were 2 to
4 cm long. The roots and shoots were separated from their
kernels with a vibrating, stainless steel gravel separator
under a shower, collected batchwise in a strainer, excess
water was shaken out, and then the material was dropped into
Il
liquid nitrogen. Packets of frozen shoots and roots (90 g
each) were wrapped in aluminum foil and stored in a Reveo
freezer (-76).
Step 2. Homogenization
One packet of seedling tissue was homogenized in a
Waring blender in 135 ml of Buffer H for 60 sec. at low
21


' .. ,1f*ACKNOWLEDGMENTS
I would like to thank Dr. Rusty J. Mans for all the
time and effort he has put forth on my behalf, both
scientifically and personally. At first I didn't know
quite.how to deal with his enthusiastic approach to prob
lems, but gradually I learned. I would like to thank
Dr. George E.''Clifford for introducing me to microbiology
and teaching me that research could be both enjoyable and
challenging. if would like to thank Dr. Ira Rosen for
introducing me to "genetics and transcription and I would
like to thank Dr. Daniel Billen for introducing me to
Dr. Rusty J. Mans. As usual, there are all the other
people and I would like to thank them too. I would like
to thank Muriel Reddish who knows everything that a
graduate student ever needs to know about the department.
My thanks to Claudia Alverez who shares everyone's
laboratory frustrations, especially those of Dr. Rusty J.
Mans. My thanks to Sharon Bryant, Bob Brooks, Gary Benson,
Carl Smith and Norm Huff for keeping me company in our
one-windowed laboratory. I would really like to thank the
N. I. H. which has kept me lean but alive during the last
five years. To my roommates, Barbara, Joe and Chantal,
"thanks, and whose turn is it to do the dishes?" And in
x i 1


110
I
n
Figure 26. A MODEL FOR POLYADENYLIC ACID
INITIATION OF TRANSCRIPTION
I- Polyadenylic acid and RNA transcribed
from the same strand of DNA.
Polyadenylic acid and RNA transcribed
from opposite strands of DNA.
II.


91
RNA,and poly(A) synthesis by the same enzyme could
be distinguished by alterations in the reaction mixture
components since: 1. ATP was a substrate for both RNA
and homopolymer synthesis, while UTP was a substrate only
for RNA synthesis (Table 6). 2. Native DNA and denatured
DNA (Table 4), poly(dAT) and poly(dAdT) (Table 14), all
satisfied the template requirement for RNA synthesis,
but only denatured DNA and poly(dAdT) satisfied the tem
plate requirement for poly(A) synthesis. 3. The NTPs
were required for RNA synthesis (Figure 11), while poly(A)
synthesis was inhibited by low NTP concentrations (Fig
ure 19). 4. Actinomycin D inhibited RNA synthesis but
stimulated poly (A) synthesis (Table 12).
The synthesis of poly(A) with ATP, but not of
poly(U) with UTP, indicated the RNA polymerase synthesized
only one homopolymer. Synthesis of homopolymer required
nucleic acid (Table 4), and the nucleic acid was utilized
as a template and not as a primer (Table 14). Therefore,
poly(dT) regions must serve as templates to synthesize
poly(A) Since Watson-Crick base pairing would require
a poly(dT) template for poly(A) synthesis on DNA, as well
as on synthetic oligomers, DNA denaturation must increase
the exposed poly(dT) regions and provide the required
template for poly(A) synthesis.


RESULTS
RNA Polymerase Purification
Utilizing the RNA polymerase .purification procedure
described in METHODS, RNA polymerase was purified 200-fold
over the 200,000 x g supernatant activity, or 1,000-fold
over that, present in the tissue homogenate. The results
of several RNA polymerase purifications are summarized in
Table 1. The steps in purification correspond to those
in Figure 1. In 10 h, 2 mg of RNA polymerase were purified
from a 90 g packet of maize roots and shoots, with a re
covery from 55 percent to 69 percent of the initial AMP
incorporating activity present in the 200,000 x g super
natant. Variability in protein and activity of the 200,000
x g supernatant probably resulted from variability in
homogenization with the Waring blender. Activity detected
in the 50 percent (NH^^SO^ precipitate was variable
(Table 1), resulting from the presence of nucleases and
variable amounts of salt in the resuspended protein. After
salt equilibration on sephadex G50, assays of polymerase
activity were less variable.
Both gradient and batch elution of DEAE-cellulose
resulted in highly active RNA polymerase. Gradient-eluted
RNA polymerase had a higher peak specific activity (104 nmoles
31


KEY TO ABBREVIATIONS (continued)
RNA
ribonucleic acid
rRNA
ribosomal ribonucleic acid
S
sedimentation coefficient
ssc
standard saline citrate
SDS
sodium dodecyl sulfate
TCA
trichloroacetic acid
tris
tris (hydroxymethyl)aminomethane
tRNA
transfer ribonucleic acid
UTP
uridine triphosphate
UMP
uridine monophosphate
50% ASP
50% ammonium sulfate precipitate
x


104
REFERENCES (continued)
69. Benson, R. H., and R. J. Mans (1972).
427abs.
70. Richardson, J. P. (1966). J. Mol. Biol.
71. Jacobson, A., and D. Gillespie (1970).
Harb. Symp. Quant. Biol., 35:85.
72. Summers, W. C., and W. Szybalski (1968)
34:9.
Fed. Proa, 31
, 21:83.
Cold Spring
Virology,
73.Albert, B. M., and L. Frey (1970). Nature, 227:1313.


Ill
Properties of Site 2
'synthesizes poly (A) and RNA, depending upon
the assay substrate and template. It binds selectively
to poly(dT) regions of denatured DNA and when synthesizing
2+
poly(A) has a strong preference for Mn as a cofactor.
In the presence of ATP it will reiteratively tran
scribe the short poly(dT) template and synthesize long
poly(A) sequences. In the presence of the NTPs, it will
synthesize a short poly(A) sequence and then begin
transcribing RNA. At low NTP concentrations, site 2
begins transcribing into the DNA and is inhibited. It
is inhibited by a-amanitin, but resistant to actinomycin
D wnen sy'Ci'iasj.i.mg pOj_y \1\) .
This model for initiation of transcription on
double stranded DNA is based upon data with denatured
DNA. The presence of a mechanism for selectively de
naturing the initiation region must, therefore, be
present iri vivo.
It is suggested the poly(dT)-rich template region
of the denatured DNA may be in a circular helix, permit
ting reiterative transcription and poly(A) synthesis
to occur.


102
REFERENCES (continued)
34.Teng, C. S., C. T. Teng and V. G. Allfrey (1971). J.
Biol. Chem., 246:3597.
35.Darnell, J. E. (1968). Bacteriol. Rev., 32:262.
36. Shearer, R., and B. J. McCarthy (1967). Biochem. J.,
6:283.
37. Blatti, S. P., personal communication.
38. Chambn, P., F. Gissinger, J. L. Mandel, C. Kedinger,
M. Gniazdowski and M. Maihlac (1970). Cold Spring
Harb Symp. Quant. Biol., 35:693.
39. Kedinger, C. P. Nuret and P. Chambn (1971). FEBS Lett.,
1_5: i69 .
40. Huang, R. C., N. Maheshwari and J. Bonner (1960).
Biochem. Biophys. Res. Comm., 3:6.
41. Bonner, J., and R. C. Huang (1966). Biochem. Biophys.
Res. Comm.. 22:211.
42. Horgen, P. A., and J. L. Key (1972). Plant Physiology,
49:13.
43. Mans, R. J., and G. D. Novelli (1964). Biochem. Bio-
phys. Acta., 91:186.
44. Stout, E. M., and R. J. Mans, Plant Physiology, 43:405.
45. Benson, R. H. (1971). Plant Physiology, 47:36abs.
46. Chamberlin, M. and P. Berg (1964). J. Mol. Biol., 8:
708.
47. Strain, G. C., K. P. Mullinix and L. Bogorad (1971).
Proc. Nat. Acad. Sci., 68:2647.
48. Edmonds, M. and R. Abrams (1962). J. Biol. Chem.,
237:2636.
49. Klemperer, H. G. (1965). Biochem. Biophys. Acta., 95:
251.
50. Mans, R. J., and T. J. Walter (1971). Biochem. Biophys.
Acta., 247:113.


8
operons (20,21). CAP and cyclic AMP were bound to the
lactose repressor protein and catalyzed its dissociation
from the operator gene (21). The results with CAP raised the
possibility that transcription of many prokaryotic and
eukaryotic genes requires the action of an additional
positive control element. The requirement for cyclic AMP
linked transcriptional control to hormone action (22).
B. Subtilis RMA Polymerase and Sporulation
The sporulation of B. subtilis resulted in changes in
the template specificity of the RNA polymerase. These
changes were associated with a decrease in transcription
of vegetative genes and the expression of new sporulation
specific genes (23,24). The sporulating cell contained
an RNA polymerase with a 3 subunit alteration such that it
could not utilize vegetative sigma factor (25). This ir
reversible change in cell phenotype resembled differentia
tion, since once sporulation began, the cell was committed
and could not return to vegetative growth except through the
sporulation stage.
Eukaryotic Transcription
General Background
While eukaryotic RNA polymerase activity was detected
as early as 1960 by Weiss (13), initial progress at


PURIFICATION OF A EUKARYOTIC
RNA POLYMERASE II THAT SYNTHESIZES
POLYADENYLIC ACID
By
ROBERT HENRY BENSON
A DISSERTATION PRESENTED TO THE GRADUATE
COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
IS 72

This dissertation is dedicated to my parents,
MaryAnn L. and Henry E. Benson, for their end
less encouragement during my 25 years of
education.

' .. ,1f*ACKNOWLEDGMENTS
I would like to thank Dr. Rusty J. Mans for all the
time and effort he has put forth on my behalf, both
scientifically and personally. At first I didn't know
quite.how to deal with his enthusiastic approach to prob
lems, but gradually I learned. I would like to thank
Dr. George E.''Clifford for introducing me to microbiology
and teaching me that research could be both enjoyable and
challenging. if would like to thank Dr. Ira Rosen for
introducing me to "genetics and transcription and I would
like to thank Dr. Daniel Billen for introducing me to
Dr. Rusty J. Mans. As usual, there are all the other
people and I would like to thank them too. I would like
to thank Muriel Reddish who knows everything that a
graduate student ever needs to know about the department.
My thanks to Claudia Alverez who shares everyone's
laboratory frustrations, especially those of Dr. Rusty J.
Mans. My thanks to Sharon Bryant, Bob Brooks, Gary Benson,
Carl Smith and Norm Huff for keeping me company in our
one-windowed laboratory. I would really like to thank the
N. I. H. which has kept me lean but alive during the last
five years. To my roommates, Barbara, Joe and Chantal,
"thanks, and whose turn is it to do the dishes?" And in
x i 1

conclusion, I would like to thank John Henry Colson and
Bernardine, for keeping me company during all my nights
in the laboratory repeating experiments and typing endless
drafts of this dissertation.
iv

TABLE OF CONTENTS
Acknowledgments iii
List of Tables vi
List of Figures vii
Key to Abbreviations ix
Abstract xi
Introduction 1
Literature Review 4
Methods and Materials. . 21
Results 31
Discussion 36
Conclusion 99
References 100
Appendix A 105
Appendix B 109
Biographical Sketch 112

LIST OF TABLES
1. STEPS IN RNA POLYMERASE PURIFICATION 32
2. STORAGE AND FREEZE-THAW LABILITY OF RNA
POLYMERASE 40
3. SALT PRECIPITATION OF RNA POLYMERASE 42
4. ASSAY REQUIREMENTS OF RNA POLYMERASE 44
5. NEAREST NEIGHBOR FREQUENCY ANALYSIS OF RNA
PRODUCT SYNTHESIZED WITH DENATURED DNA. 59
6. AMP AND UMP INCORPORATION BY RNA POLYMERASE . .65
7. INHIBITION OF POLYADENYLIC ACID SYNTHESIS BY
NUCLEOTIDES . 67
8. EFFECT OF DELAYED ADDITION OF NTPs ON POLYADENYLIC
ACID SYNTHESIS. .68
9. EFFECT OF HEATING' AND SDS ON RNA POLYMERASE
PRODUCTS 71
10. NEAREST NEIGHBOR FREQUENCY ANALYSIS OF POLY
ADENYLIC ACID PRODUCT .72
11. AVERAGE CHAIN LENGTH OF POLYADENYLIC ACID 73
12. INHIBITOR SENSITIVITY OF RNA POLYMERASE 76
13. COMPARISON OF CALF THYMUS AND MAIZE DNA AS
TEMPLATES FOR RNA POLYMERASE 82
14. RNA POLYMERASE ACTIVITY WITH SYNTHETIC TEMPLATES. .84
vi

LIST OF FIGURES
1. PURIFICATION PROCEDURE FOR RNA POLYMERASE 22
2. DEAE-CELLULOSE GRADIENT ELUTION ASSEMBLE ..... 25
3. DEAE-CELLULOSE ELUTION PROFILE' 34
4. RNA POLYMERASE ACTIVITY IN DEAE-CELLULOSE
FRACTIONS 35
5. POLYACRYLAMIDE GEL ELECTROPHORESIS OF NATIVE RNA
POLYMERASE 37
6. POLYACRYLAMIDE GEL ELECTROPHORESIS OF DENATURED
RNA POLYMERASE .38
7. DNA TITRATIONS WITH MAGNESIUM AS COFACTOR 46
8. DNA TITRATIONS WITH MANGANESE AS COFACTOR 47
9. MAGNESIUM TITRATION WITH NATIVE AND DENATURED DNA. 49
10. MANGANESE TITRATION WITH NATIVE AND DENATURED DNA. 50
11. NTP TITRATION OF. RNA POLYMERASE. 51
12. ATP TITRATION OF RNA POLYMERASE 53
13. LINEWEAVER-BURKE PLOTS OF ATP TITRATIONS 54
14. AMMONIUM SULFATE TITRATION OF RNA POLYMERASE ... 55
15. RNA POLYMERASE ACTIVITY AS A FUNCTION OF ENZYME
CONCENTRATION. . 57
16. RATE OF AMP INCORPORATION AS A FUNCTION OF TIME. 58
17. DNA TITRATIONS WITH MAGNESIUM IN THE ABSENCE OF
NTPs 62
18. DNA TITRATIONS WITH MANGANESE IN THE ABSENCE OF
NTPS 63
vii

LIST OF FIGURES (continued)
19. NTP TITRATION WITH DENATURED DNA 66
20. POLYADENYLIC ACID SYNTHESIS AS A FUNCTION OF
ENZYME CONCENTRATION. 70
21. ALPHA-AMANITIN TITRATION OF RNA POLYMERASE. ... 78
22. ACTINOMYCIN D TITRATION OF RNA POLYMERASE .... 79
23. RIBONUCLEASE SENSITIVITY OF DENATURED DNA-DEPENDENT
PRODUCTS. 81
24. AMP INCORPORATION AS A FUNCTION OF ATP SPECIFIC
ACTIVITY 106
25. RATE OF AMF INCORPORATION AS A FUNCTION OF ATP
CONCENTRATION 108
26. A MODEL FOR POLYADENYLIC ACID INITIATION OF
TRANSCRIPTION 110
viii

KEY TO ABBREVIATIONS
A
260
ATP
AMP
BSA
CAP
cpm
CTP
d
DMSO
dpm
DEAE
DNA
GTP
g
h
HrRNA
NTPs
POPOP
PPO
poly(A)
poly(dAT)
poly(U)
poly (cLAdT)
absorbancy at 260 run
adenosine triphosphate
adenosine monophosphate
bovine serum albumin
catabolite gene-activator protein
counts per minute
cytidine triphosphate
dalton
dimethyl sulfoxide
disintegrations per minute
diethylaminoethane
deoxyribonucleic acid
guanosine triphosphate
gravity
hour
heterogeneous nuclear ribonucleic acid
GTP, CTP, UTP
1.4-bis-[2-(4-methyl-5-phenyloxazolyl)]-
benzene
2.5-diphenyloxazole
polyadenylic acid
alternating copolymers of deoxyadenylic
and deoxythymidylic acid
polyuridylic acid
homopolymers of deoxyadenylic and
deoxythymidylic acid-
ix

KEY TO ABBREVIATIONS (continued)
RNA
ribonucleic acid
rRNA
ribosomal ribonucleic acid
S
sedimentation coefficient
ssc
standard saline citrate
SDS
sodium dodecyl sulfate
TCA
trichloroacetic acid
tris
tris (hydroxymethyl)aminomethane
tRNA
transfer ribonucleic acid
UTP
uridine triphosphate
UMP
uridine monophosphate
50% ASP
50% ammonium sulfate precipitate
x

Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
PURIFICATION OF A EUKARYOTIC RNA POLYMERASE II
THAT SYNTHESIZES POLYADENYLIC ACID
By
Robert Henry Benson
December, IS72
Chairman: Rusty J. Mans, Ph.D.
Major Department: Immunology and Medical Microbiology
The in vitro catalytic activities of DNA-dependent
RNA polymerase II, isolated and purified from rapidly
growing 5-day-old maize seedlings (WF9 x Bear 38, waxy),
have been examined. An enzyme purification procedure is
described utilizing homogenization, differential centri
fugation, salt precipitation and ion exchange chromoto-
graphy that resulted in 1,000-fold purification with
70 percent recovery in 6 to 10 hours. The enzyme had a
specific activity greater than 100 nmoles AMP/mg/20 min.
at 30, and contained one major band and several minor
bands on polyacrylamide gel electrophoresis. Incorpora
tion of AMP into acid-insoluble material required DNA,
4 nucleosidetriphosphates: GTP, CTP, UTP, ATP (NTPs),
2+ 2+
and metal, either Mn or Mg Alkaline hydrolysis of
xi

product, synthesized with [a-32p]ATP, NTPs, denatured DNA
2+
and Mn resulted in these nearest neighbor frequencies:
CpA, 0.25; ApA, 0.44; GpA, 0.18; UpA, 0.13. The require
ments for incorporation of a labeled nucleotide into acid-
insoluble material and the nearest neighbor frequency
indicate that RNA was synthesized. In the presence of
labeled ATP alone, the ApA frequency of alkaline-hydrolyzed
product was 0.90, and the AMP/adenosine ration was greater
than 100, indicating polyadenylic acid [poly(A)] was syn
thesized. Since UTP was incorporated in the presence but
not in the absence of NTPs, no poly(U) was synthesized,
suggesting that poly(A) may be the only homopolymer
accumulated. Poly (A) synthesis was 90 percent inhibited
by 0.025 mM NTPs, whereas both RNA and poly(A) were syn
thesized with 1.0 mM (or greater) NTPs, suggesting the
mode of poly(A) synthesis differs as a function of the nu
cleotides present, but all products synthesized, were acid-
insoluble after heating (100) for 5 min. in 1 percent SDS,
indicating that product-protein and product-DNA complexes
were not responsible for acid-insolubility. RNA and
poly(A) synthesis was inhibited by 1 yM a-amanitin,
indicating that both products were synthesized by RNA
polymerase II. Poly(A) synthesis was supported by poly(dAdT),
but not by poly(dAT), suggesting a poly(dT) template was
utilized for poly(A) synthesis. Actinomycin D (7 yM)
inhibited RNA synthesis 87 percent on denatured calf thymus
Xll

DNA, but stimulated poly(A) synthesis 28 percent, again
suggesting poly(dT) regions of DNA were utilized as a
template for poly(A) synthesis. Neither RNA nor poly(A)
synthesis was inhibited by cordycepin (0.26 mM) or
rifampicin (50 yg/ml), therefore eliminating the presence
of maize NTP: exotransferase and bacterial RNA polymerase
activity. Since early product (accumulated at a high rate
of AMP incorporation) was resistant to pancreatic ribo-
nuclease, whereas, late product (accumulated at a lower
rate of AMP accumulation) was sensitive to RNase digestion,
poly(A) synthesis apparently preceded RNA synthesis. A
model is presented which requires that poly(A) synthesis
preceda RNA synthesis in the same enzyme-template complex
during the initiation of transcription in eukaryotes.
xiii

INTRODUCTION
Macromolecular nucleic acid metabolism depends upon
two classes of enzymes: the polymerases which assemble
the polymers from the nucleoside or deoxynucleoside tri
phosphates, and the nucleases which break down the polymers.
Among the polymerases there are four types which are known
to utilize a nucleic acid as a template to synthesize a
new polymer. These are DNA-dependent DNA polymerase
(EC 2.7.7.7), DNA-dependent RNA polymerase (EC 2.7.7.6),
RNA-dependent RNA polymerase and RNA-dependent DNA. poly
merase. Of primary importance in phenotypic expression of
genetic information are the DNA-dependent RNA polymerases.
These enzymes are responsible for the synthesis of messenger
RNA,the template for sequencing amino acids, and for the
synthesis of ribosomal and transfer RNAs, needed for trans
lation of the messenger RNA into protein. Control of the
synthesis of these RNAs seems to be primarily at the level
of initiation of transcription.
Transcription may be studied in vivo or in vitro.
The in vitro approach to transcription requires a purified
RNA polymerase. Once the enzyme is purified, specific
properties can ten be determined, such as the assay
1

2
requirements and product characteristics. Following
this initial evaluation, transcriptional control functions,
such as template specificity or the presence or absence
of initiation and termination factors, can be determined.
The DNA-dependent RNA polymerases all require a
DNA template, the four nucleoside triphosphates: ATP,
UTP, GTP and CTP, and metal during the synthesis of RNA
and concomitant liberation of pyrophosphate. The RNA
polymer is synthesized in the 5' to 3' direction. The
new nucleotide is linked via a phosphodiester linkage to
the 3' hydroxyl of the growing RNA chain. As the RNA is
synthesized in the 5' to 3' direction, the template DNA
is transcribed in the 3' to 5' direction. RNA polymerase
activity can be evaluated separately in four operational
steps: binding, initiation, elongation and termination.
Step one is the binding of the RNA polymerase to the DNA
template. Step two is the initiation of an RNA chain
via synthesis of the first phosphodiester linkage. Step
three is the elongation of the new RNA chain. Step four
is the termination of RNA synthesis. Release of the RNA
chain may occur immediately or it may be delayed.
Experimental evaluation of the initial steps,
binding to the DNA template and initiation of RNA syn
thesis, requires an enzyme of high specific activity.
This high activity is required to experimentally detect

3
the initial synthetic activity of the RNA polymerase
and to minimize the influence of any other enzymatic
activities present. In addition, experimental manipu
lation of the enzyme during purification should be kept
minimal, to reduce the probability of enzyme degeneration
and, therefore, to preserve in vivo transcriptional
activity. The experimental approach in this study was
to purify the RNA polymerase from maize seedlings, to
determine its requirements for product accumulation and
to identify the products. RNA polymerase was purified
by centrifugation, (NH^)2SC>4 precipitation and ion ex
change column chromotography in that order. From its
assay requirements and inhibitor sensitivities it was
identified as SNA polymerase type II. In addition to its
known catalysis of RNA synthesis, this eukaryotic RNA
polymerase catalyes polyadenylic acid synthesis. The
polymerase products were characterized by sensitivity to
heat, SDS and pancreatic ribonuclease, and by nearest
neighbor frequency analysis. The possible involvement of
poly(A) synthesis as a mechanism of initiation of tran
scription is suggested and discussed.

LITERATURE REVIEW
Approaches to Transcription
Guides to Literature
There are several avenues into the literature of
transcription; for current research, the symposia (1,2,3)
and reviews (4,5,6) are best, whereas for a more historical
perspective, there are collected papers (7) and introductory
texts (8,9,10) .
Systems Studied
Essentially every level of life from the smallest
viruses to the largest eukaryotes has had, or is having,
its transcriptional processes studied. The basic enzymology
has been done in bacterial systems, principally Escherichia
coli and Bacillus subtilis and their phages (11,12).
Although RNA polymerase was first detected in a eukaryote
by Weiss in 1960 (13), the bacterial viruses and their
nucleic acids proved most useful in elucidating transcription
in prokaryotic systems (11,14). The major difficulties en
countered in studying the eukaryotic systems were the initial
low activity of the eukaryotic RNA polymerases and the dif
ficulty in getting the enzyme DNA-dependent (15). These
4

5
problems have been solved and the study of in vitro
transcription in eukaryotes is rapidly advancing.
Transcription as a subject may be broken into two
areas of investigation: first, the prokaryotes and their
virases that have served as model systems due to the ease
of acquiring enzyme and defined DNA templates; second, the
eukaryotes that have complex chromosomes and multiple
polymerase.systems producing the intricate specialized
tissues of plants and animals. The bacterial systems will
be illustrated by.the E. coli and B. subtilis systems and
the eukaryotes by the calf thymus and maize systems.
Bacterial Transcription
Significant Concepts
There -are extensive reviews of transcription available
(1,2,4,5,6). I would like to draw from these systems to
illustrate four concepts which have recently been uncovered
in bacterial transcription.
1. RNA polymerase and associated factors have
the ability to initiate and terminate tran
scription at specific sequences on the
chromosome.
. Alteration of the RNA polymerase, either
through viral infection or sporulation,
results in a change in the transcription
specificity of the RNA polymerase.
2

6
3. RNA polymerase may be the site of hormone
action via specific protein factors such
as CAP .(catabolite gene-activator protein)
which are influenced by hormone-controlled
cyclic AMP.
4. Alteration of the RNA polymerase transcription
specificity can result in irreversible
changes in cell phenotype, closely
resembling differentiation in eukaryotes.
These four concepts illustrate the potential importance of
a better understanding of the RNA polymerases in both pro
karyotes and in eukaryotes.
E. Coli RNA Polymerase and Sigma Factor
E. coli RNA polymerase contains four types of subunits:
g' (155-165,000 d), 3 (145-155,000 d), a (85-95,000 d), and
a (39-41,000 d). A fifth subunit, u (10,000 d), is some
times found with the RNA polymerase although it is not
required for RNA polymerase function (4). Together these
subunits are arranged as holenzyme (8'Ba a), which retains
2
the ability to asymmetrically transcribe T4 DNA, or as core
enzyme (g'Ba ), which cannot asymmetrically transcribe T4
2
DNA (11) The presence or absence of sigma (a) determines
the initiation specificity of the RNA polymerase on T4 DNA.
Another factor, rho (p), although not bound to the RNA poly
merase, when present with the enzyme resulted in one type
of specific termination of RNA synthesis (15) Two other
sites also resulted in chain termination. The RNA poly
merase, without rho, also recognized sequences in the DNA
transcribed as UAA (ochre) and UAG (amber) and terminated

7
RNA chains (15) .
In response to infection by coiiphage T4, E. coli
RNA polymerase transcribed early T4 RNA. After production
of the gene 55 protein, translated from the early RNA, the
synthesis of delayed early RNA was specifically initiated
(16). Travers (17) isolated the protein and called it a
T4 sigma-like factor which caused specific asymmetric
initiation of T4 DNA at the site of delayed early RNA. This
isolation of a factor which directed delayed early RNA
synthesis demonstrated for the first time that RNA poly
merase could acquire a new initiation specificity (16,17).
One minute after infection by phage T4, the host RNA poly
merase no longer synthesized early T4 RNA (18). This
change was apparently caused by alteration of the a sub
unit of the host RNA polymerase through adenylation with
5'AMP (19). Therefore, viral infection initiated changes
in the transcription machinery which caused a specific
alteration of the initiation sites for RNA synthesis.
These changes included alteration of the existing host RNA
polymerase and the synthesis of a viral protein to replace
the host sigma factor.
E. Coli RNA Polymerase and CAP
RNA polymerase control in coli illustrates the
possible importance of transcription as a mechanism for
hormone action. Cyclic AMP, together with CAP, was required
for maximum expression of the lactose and other inducible

8
operons (20,21). CAP and cyclic AMP were bound to the
lactose repressor protein and catalyzed its dissociation
from the operator gene (21). The results with CAP raised the
possibility that transcription of many prokaryotic and
eukaryotic genes requires the action of an additional
positive control element. The requirement for cyclic AMP
linked transcriptional control to hormone action (22).
B. Subtilis RMA Polymerase and Sporulation
The sporulation of B. subtilis resulted in changes in
the template specificity of the RNA polymerase. These
changes were associated with a decrease in transcription
of vegetative genes and the expression of new sporulation
specific genes (23,24). The sporulating cell contained
an RNA polymerase with a 3 subunit alteration such that it
could not utilize vegetative sigma factor (25). This ir
reversible change in cell phenotype resembled differentia
tion, since once sporulation began, the cell was committed
and could not return to vegetative growth except through the
sporulation stage.
Eukaryotic Transcription
General Background
While eukaryotic RNA polymerase activity was detected
as early as 1960 by Weiss (13), initial progress at

9
purifisaatlo-x^WAS. vex^. slow. This was a. result of the
initial |.o^'avti^ty :of the RNA polymerase in eukaryotic
tissues, of fcfej&f&iiwf-i-culty encountered in freeing the enzyme
of contaminating 'DNA and of the instability of the enzyme
during purification (26) These problems were overcome and
soluble DNA-dependent RNA polymerases were purified from
eukaryotic tissues (27,28,29,30). In 1970, Roeder and
Rutter (31) first detected the presence of two types of
RNA polymerase--5:' Hype I was a nucleolar RNA polymerase,
insensitive to a-amanitin, that synthesized a product which
competitively-..hybridized with r-RNA but not with Hn-RNA.
Type II was a nupl^oplasmic, a-amanitin sensitive RNA poly
merase that synthesized a product that competitively
hybridized with Hn-RNA but not with r-RNA (32). This was
the first time that specific RNA polymerases were shown
to be localized inside a eukaryotic cell and to be re
sponsible for specific classes of eukaryotic RNA. Since
then it has been shown that the nucleus, the nucleolus,
and the cellular organelles have unique transcriptional
systems (15).
Eukaryotes have a complex chromosomal structure
involving nucleic acids, histones, and acidic proteins.
The relationship between the histones and differentiation
is. uncertain, although histones are believed to be inti
mately involved 'in gene selection (33). There are two
major approaches to how this interaction might occur.

10
First, transcription may be activated by removal of histones,
somewhat analogous to removal of the repressor on the lactose
operon. Second, RNA polymerase and specific initiation
factors initiate transcription by opening the genes for
transcription, with histone removal occurring as the RNA
polymerase precedes (15). Accordingly, either the histones
or the RNA polymerases may function to control the specificity
of transcription, or a combination of both systems.
Acidic nuclear proteins are of interest to transcription
because of their physical properties and location (34).
Since the RNA polymerases are themselves acidic nuclear
protein complexes, some of the acidic proteins may be sub
units of the RNA polymerases or of the other polymerases.
The acidic nuclear proteins may contain factors that function
along with the histones for gene selection and control of
transcription.
Transcription Products
There are at least three classes of RNA produced in
all cells: rapidly labeled RNA including HnRNA and mRNA;
stable RNA or GC-rich RNA which is rRNA; and soluble or
tRNA. These three classes of RNAs constitute the bulk of
the transcriptional products in all cells. Following
transcription, the RNA is often processed by specific
systems which selectively degrade the gene product into
the functionally active form. The processing of ribosomal
T

11
RNA is the best studied. The ribosomal genes are
sequestered.- in the nucleolus and are transcribed as a unit
into a singde ?40-45 S precursor RNA molecule. This RNA is
pxocessed by a series of post-transcriptional cleavage steps
to give 18 S and 25 S RNAs found in mature amphibian oocyte
ribosomes (35). Heterogeneous RNA is found in the nucleo
plasm and has a DNA-like base composition. A large fraction
of this rapidly labeled RNA never leaves the nucleus and
may be involved with regulation at the level of transcription
(36). Both rRNA and tRNA are stable and constitute the
bulk of the RNA contained in a cell at any one time, while
mcvst of the- rRNA rapidly turns over.
RNA Polymerases
Types
Type I RNA polymerase is localized in the nucleolus
and synthesizes GC-rich RNA that competitively hybridizes
with ribosomal RNA [32). Its polymerizing activity is
resistant to ct-amanitin, it is highly sensitive to actinomycin
D and it is refractory to rifampicin (32). On DEAE-sephadex
it is the first RNA polymerase eluted by a linear ammonium
sulfate gradient, generally around 0.2 M (NH^^SO^. The
type I RNA polymerase has a preference for magnesium and
low salt concentrations (0.04-0.07 M (NH^^SO^). The type I
RNA polymerase is very unstable and therefore difficult to
purify (37).

12
The type II RNA polymerase is a nucleoplasmic RNA
polymerase that synthesizes a product which competitively
hybridizes with HnRNA. (32). Its activity is sensitive
to a-amanitin and it is the second RNA polymerase eluted
by a linear (NH^^SO^ gradient from DEAE-sephadex, generally
around 0.3 M salt. The type II enzyme exhibits more
activity with manganese, rather than with magnesium, and
is most active at high salt concentrations (0.9-0.12 M
(NH^^SO^) (32). It is more stable than the type I RNA
polymerase, but is inactivated easily, particularly during
salt precipitation.
Type.Ill RNA polymerase is a nucleoplasmic RNA
polymerase, eluted third on DEAE-sephadex chromotograph,
generally around 0.35 M (NH4)2SC>4 (32). The small amount
of type III enzyme activity present in extracts is often
undetected. The type III RNA polymerase is resistant to
a-amanitin, prefers manganese and has a broad salt optimum
(0-0.2 M (NH4)2SC>4) (32). It is the least studied of the
nuclear RNA polymerases and it has been proposed that it may
synthesize tRNA (32) .
The type IV RNA polymerase, refers to RNA polymerase
activity detected in cellular organelles, specifically
chloroplasts and mitochondria. Little is known of these
enzymes due to the difficulty in extracting the quantity of
material necessary for enzyme purification, and to the
difficulty in removing the other contaminating RNA polymerases.
4

13
Most studies of organelle transcription therefore utilized
in vivo ..labeling and experimentation.
Structure
RNA polymerase II has been purified from calf thymus,
rat liver, sea urchin, yeast, mouse embryo cells, Hela
cells, xenopus and maize (1,5,27). The subunit structure
of the type II RNA polymerase from calf thymus was evalu
ated on SDS-polyacrylamide gel electrophoresis (38) These
subunits had molecular weights of 215,000, 185,000 and
150,000 and were designated Bl, B2, and B3. Gel densitom
etry indicated a ratio of 1:1:2 between the gel bands
Bl:B2:B3 (39). These subunits apparently came from two
subsets of RNA polymerase II. The first subtype gave
bands Bl and B3, the second subtype gave bands B2 and B3.
There were also 3 smaller subunits of molecular weights
20,000, 30,000 and 40,000. The large subunits Bl, B2 and
B3 appeared to be similar to the 0 and 3' subunits of the
E. coli RNA polymerase. It has been suggested that cr-amanitin
inhibits the type II RNA polymerase by binding to subunit
B3.
Maize RNA Polymerase
Maize RNA polymerase was one of the first eukaryotic
RNA polymerases solubilized (27). Other eukaryotic RNA
polymerases were solubilized from animal tissues (1);
however, other plant RNA polymerases were studied primarily

14
.using plant chromatin or crude salt fractionated supernatants
(40,41,42). ENA polymerase activity was first detected in
the soluble fraction of a French pressure cell extract in
1964 by Mans and Novell! (43) The RNA polymerase in
this extract was further purified by DEAE-cellulose chrorao-
tography using a linear Tris-HCl gradient (Stout and Mans
1967) (27). The average specific activity eluted was 4.06
nmoles AMP/mg at 10 min. This eluted RNA polymerase would
utilize either native or denatured DNA as a template
equally well, although at low DNA levels (10 ug DNA/ml) dena
tured DNA was eight times as effective as native DNA (44).
Denatured calf thymus DNA was more efficient as a template
than denatured maize DNA; however, the calf thymus DNA had
a much greater hyperchromicity than maize DNA, 17 percent
vs 8.3 percent (44). The RNA polymerase required all four
nucleoside triphosphates, a bivalent metal ion, and DNA to
14
incorporate [8- C]ATP into acid-insoluble material (27).
The metal could be either magnesium (25 mM) or manganese
(5mM) If [ct-32p]UTP or [ct-32p]ATP were used as labeled
substrate, the nearest neighbor frequency indicated RNA
containing all four nucleosidemonophosphates had been syn
thesized (27) The reaction was inhibited by actinomycin D,
pyrophosphate and DNase (43). The product synthesized on
native DNA was greater than 90 percent digested by pancreatic
ribonuclease, while on denatured DNA this decreased to 73
percent (27). On sucrose density gradients the products
1

15
were 14-16 S, which corresponded to the distribution of
the denatured DNA template (44) This distribution was
that expected for DNA-RNA hybrids. As expected, upon
heating the complexes disaggrated and all nucleic acids
were at the top of the gradient (4-6 S).
The RNA polymerase purified by the method of Stout
and Mans was identified as a type II nucleoplasmic RNA
polymerase by its sensitivity to a-amanitin (45) This
type II RNA polymerase did not synthesize homopolymers
such as poly(A) with denatured DNA, as many bacterial
RNA polymerases did (46), for with denatured DNA "the
formation of a homopolymer was not detected with the maize
polymerase" (44, p. 752), nor was it detected with any
other eukaryotic RNA polymerase.
A type I maize RNA polymerase was reported by Strain
et al. (47) Maize leaves were used as crude material and
carried through DEAE-cellulose chromotography. The type I
RNA polymerase eluted at 0.08 M (NH^^SO^ (47). Strain,
et al. also detected two overlapping peaks of activity in
the type II RNA polymerase region. One peak preferred
native DNA, the other preferred denatured DNA as measured
by total AMP incorporated (47) The metal requirements of
the leaf RNA polymerase indicated a preference for magnesium
over manganese, with optimums at 2 5 mM for magnesium and
8 mM for manganese (47).

16
Polyadenylic Acid
Enzymatic Synthesis
A eukaryotic polyadenylic acid polymerase was first
discovered in calf thymus by Edmonds and Abrams in 1962
(48). Its activity was inhibited by the other nucleotide
triphosphates, it required magnesium and it was particulate,
perhaps bound to the nuclear membrane. Its product was
almost pure poly(A), except for about 1 percent of the
adenylate residues which were joined to cytidylate resi
dues. The enzyme could not be freed of endogenous RNA.
Others have purified poly(A) polymerases from rat liver
(49) and from maize (50). The maize poly(A) polymerase
adds poly(A) chains to the 3' hydroxyl of primer nucleic
acids, either RNA or DNA (51).
Poly(A) synthesis has also been studied in prokaryotic
systems. E. coli RNA polymerase, with denatured calf thymus
DNA and only ATP as substrate, will synthesize poly(A)
sequences using poly(dT) regions of the DNA as template
(46). Reiteritive transcription of poly(dT) regions, each
greater than 5 nucleotidyl residues long, resulted in the
synthesis of long poly(A) chains through a repeated
utilization of the poly(dT) template by an unknown mechanism
Poly(A) synthesis required denatured DNA and was inhibited
by the addition of the other nucleoside triphosphates.
The polymerase would not lengthen added poly(A) primers (46)

17
.The E. coli RNA polymerase had a greater affinity for
denatured DNA than for native DNA (52), perhaps reflecting
a greater binding affinity for the exposed poly(dT) regions
of the denatured DNA.
Importance
From 1361 to 1968, poly(A) synthesis by prokaryotic
RNA polymerase was an unusual artifact of the assay and of
unknown significance. With the discovery of poly(A) se
quences in RNA isolated from numerous eukaryotes; vaccinia
virus cores (53), Hela cells (54,55), mouse sarcoma cells
(56), and avian myeloblastosis virus (57), poly(A) syn
thesis again became of interest. In eukaryotes only a
portion of the DNA-like nuclear RNA is transported to the
cytoplasmic polysomes. In addition, the nuclear RNA is
much larger in size than that found in the cytoplasm (53).
In studying poly(A) synthesis in vaccinia viral cores,
Kates asked, "Could poly(A) sequences in nuclear RNA
play a role in either the cleavage of RNA into smaller
pieces or in .the selective transport of certain species
to the cytoplasm?" (53, p. 752). To answer the question
of the role of poly(A), two additional questions should
first be answered. Is the poly(A) covalently attached to
the RNA, and if so, where? If attached to RNA, what
enzyme catalyzed the synthesis of the poly(A) sequence?
Finding some poly(A) attached to RNA does not imply that

18
all the poly(A) was attached, or remained attached.
Knowing the time and location of poly(A) synthesis and the
enzymes responsible would contribute significantly to an
understanding of the physiological function of poly(A)
synthesis.
Location in Vivo
Poly(A) can be isolated from the HnRNA or from the
rapidly labeled RNA isolated from polyribosomes (54,55,56).
Edmonds (54) indicated the data was consistent with the
idea that every HnRNA contained at least one poly(A) se
quence. More than one mode of poly(A) synthesis was
implicated since cordycepin (3-deoxyadenosine) suppressed
the labeling of mRNA found on Hela ribosomes while not
effecting the labeling of nuclear RNA (58). This suggested
there may be two enzymatic activities that synthesized
poly(A), one sensitive to cordycepin that synthesized
poly(A) and one insensitive to cordycepin that synthesized
HnRNA containing poly(A) sequences. Lim and Cannelakis'
(59) results with haemoglobin mRNA indicated that, at most,
it could contain 70 polypurine residues of 70 percent AMP.
Furthermore, this polypurine sequence was not at the 3'
end of the haemoglobin mRNA, since the 3' end contained
only 7 or 8 AMP residues before the first pyrimidine (60).
Therefore, the poly (A) must be at the 5' end or inside the
RNA chain, if there at all.

19
Tiie strongest evidence for poly (A) being contained
in newly synthesized RNA was that of Kates (53) Utilizing
' vacciiiiV cores incubated in vitro, under conditions of RNA
synthesis, Kates'data indicated that after mild RNase treat
ment all of the RNase resistant poly(A) sequences sediment-
ated at 4 S and had a chain length greater than 50 nucleo
tides. The association of poly(A) with RNA was not dis
rupted by heating at 100, nor by 75 percent DMSO at 80
,in the presence of cold poly(A). This indicated that, if
in fact, the poly (A) was attached to the RNA it was probably
through a covalent bond. When poly(A) was synthesized by
vaccinia cores with only ATP as the substrate, then the
poly(A) was not later attached to newly synthesized viral
RNA (53). This in vitro poly(A) synthesis continued for
only 5 min., the poly(A) was 180 nucleotides long and had
a uniform 5.8 S value. After alkaline hydrolysis and
chromotography there was one 5' tetraphosphate and one
adenosine for every 180 AMP residues (53). If synthesis
'-occurred in the presence of all 4 nucleoside triphosphates,
25-30 percent of the AMP incorporated was in poly(A). The
poly(A) was synthesized without a lag period upon addition
of the substrates, but if UMP incorporation was measured,
there was a 1.5 min. lag before incorporation began (53).
This indicated poly(A) synthesis preceded RNA synthesis.
If the poly(A) synthesized in the presence of all 4 nucleo
side triphosphates was purified and alkaline hydrolyzed,

20
there were 5 adenosine residues for every adenosine
tetraphosphate (53). Therefore, if poly(A) was the initial
sequence and contained the tetraphosphate, there were four
more sequences that were synthesized internally or at the 3'
terminis.
Based upon hybridization of poly(A) to denatured
vaccinia DNA, Kates estimated there could be greater than
25 poly(dT) sequences in the vaccinia DNA, each 180
nucleotides long (53). This wa consistent with Heaust
and Botchan (61), who stated that 10 percent of the genome
of mice consisted of AT-rich regions. The function of this
DNA was not clear, but it was probably not transcribed in
vivo (61). It was, however, known to be uniformly distri
buted among all chromosomes.
Summary
Poly(A) sequences are known to occur in vivo and in
vitro. They appear to be synthesized by two enzyme systems,
poly(A) polymerases sensitive to cordycepin, and by RNA
polymerases insensitive to cordycepin. Some poly(A) se
quences are located at the 3" hydroxyl end of RNA polymers;
however, there is evidence for both poly(A) sequences in
ternally and at the 5' end of the RNA. The importance and
functional significance of these sequences is not firmly
established.

METHODS AND MATERIALS
Methods
RNA Polymerase Purification
The purification of maize RNA polymerase has evolved
through enumerable modifications of that published initially
(27). Each step in the procedure utilized here is indicated
in Figure 1.
Step 1. Preparation of material
Grain (4-10 liters), in a plastic garbage can with a
perforated bottom, was imbibed and germinated under run
ning water (23) for five days until the shoots were 2 to
4 cm long. The roots and shoots were separated from their
kernels with a vibrating, stainless steel gravel separator
under a shower, collected batchwise in a strainer, excess
water was shaken out, and then the material was dropped into
Il
liquid nitrogen. Packets of frozen shoots and roots (90 g
each) were wrapped in aluminum foil and stored in a Reveo
freezer (-76).
Step 2. Homogenization
One packet of seedling tissue was homogenized in a
Waring blender in 135 ml of Buffer H for 60 sec. at low
21

22
SEEDLINGS
KERNELS
ROOTS AMD SHOOT
STEP 2 HOMOGENIZE
FILTER BOUND
STEP 3 1 CENTRIFUGE
PELLET
STEP 4 | 50% (NH4)2S04
SUPERNATANT
PELLET
STEP 5 SEPHADEX 650
INCLUDED VOLUME
EXCLUDED VOLUME
tsS
STEP 6 DEAE-CELLULOSE
swsske^
RNA POLYMERASE
Figure 1. PURIFICATION PROCEDURE FOR RNA POLYMERASE

23
speed and for 15 sec. at high speed. The homogenized
material had the consistency of a thick milkshake. The
homogenized material was immediately filtered through four
layers of cheesecloth, through a layer of miracloth and
into a chilled Erlenmeyer flask. Material retained in the
filters was discarded.
Step 3. High speed centrifugation
The filtered homogenate containing 20 to 40 percent
of the protein present in the shoots and roots was centri
fuged for 60 min. in a Ti50 rotor at 200,000 x g at 0.
The supernatant fraction was decanted into a chilled
graduated cylinder through a layer of miracloth, which re
tained the lipid layer accumulated at the top of the centri
fuge tube. The supernatant fraction contained approximately
50 percent of the protein present in the filtered homogenate.
Step 4. Ammonium sulfate precipitation
To the high speed supernatant an equal volume of
saturated (NH^^SO^ was added slowly with continuous and
gentle stirring in an ice-jacketed beaker. After 30 min.
of stirring the resulting precipitate was collected by
centrifugation (10,000 x g, 10 min., 0) and resuspended
in a minimal volume of Buffer R (approximately 15 ml).

24
Step 5. Salt equilibration on sephadex
Equilibration in Buffer E was accomplished by passage
erf -the "'resuspended precipitate through a sephadex G50
column(2.5 x 11 cm) equilibrated with Buffer E. The ex
cluded material was diluted to 25 ml with Buffer E for
absorption to DEAE-cellulose.
Step 6. DEAE-cellulose chromotography
The excluded volume eluted from sephadex G50
chromotography was loaded (1 ml/min.) onto a DEAE-cellulose
(see materials) column (2.5 i.d. x 11 cm) equilibrated with
Buffer E. The loaded column was washed with 60 ml of
- -,vt
Buffer E and the flow rate was decreased to 0.25 ml/min.
A 60 ml (NH^)2S0^ gradient (0.20 to 1.0 M) was begun
immediately after passage of the 60 ml of Buffer E.
Column eluates were monitored and recorded by a continuous
flow ultraviolet-monitoring system (Gilford spectrophoto
meter and a Honeywell recorder, Figure 2). Eluted fractions
were collected directly from the flowcell into glass vials
(2.5 ml/vial) and frozen in liquid nitrogen.
Polymerase Assay
RNA polymerase was assayed in a 0.10 ml standard
reaction mixture containing: 10 ymoles Tris-Hcl, pH 7.6 @
25; 0.25 ymoles each UTP, CTP, GTP, (sodium salts);
0.10 ymoles [8--^C]ATP (specific activity 1.7 to 4.5 yC/ymole) ;
1.0 ymole MgC^; 1*0 ymole 2-mercaptoethanol; 8 ymoles

GRADIENT
SOLUTIONS
}>
o
rn
m
c
>
o
H
o
PI
o
r
C
o
r?
A39
<
2
n
l
VIALS
ICE
BUCKET
V
TO
LIQUID
NITROGEN
Figure 2. DEAE-CELLULOSE GRADIENT ELUTION ASSEMBLE
to
<_n
4

26
(NH^^SO^; 0.37 calf thymus DNA; 5 yg BSA and enzyme
as indicated (27). An assay mixture was prepared immediately
before use and held on ice until the reaction was initiated
by the addition of enzyme to the mixture. The reaction
tube was immersed in a 30 water bath for 20 min. and acid-
insoluble radioactivity was determined by a modification of
a procedure previously described (27). The reaction was
terminated by pipetting the mixture onto a 3 MM filter
paper disk. The reaction mixture was absorbed into the
disk for 10 sec. under a heat lamp and precipitated by
immersion in cold 10 percent TCA containing 2 mM sodium
pyrophosphate. As many as 40 disks were then extracted
five times with 100 ml of the TCA solution (5 min. each
time), once with 100 ml ethanol-ether (1:1), and once with
100 ml ether for 3 to 5 min. All extractions were per
formed at room temperature. The disks were then dried
and counted in a scintillation solution containing PPO,
POPOP and reagent grade toluene. Disks which received
reaction mixture containing no enzyme averaged 15 cpm
above machine background. The wash procedure reduced the
cpm from approximately 400,000 cpm/disk to 35 cpm/disk
for the no enzyme control. Machine counting efficiency
was 0.75 cpm/dpm. One unit of RNA polymerase activity is
defined as 1 nmole AMP incorporated into acid-insoluble
material under standard reaction mixture conditions in
20 min.

27
Protein Determination
Protein was measured on samples precipitated with
10 percent trichloroacetic acid by the method of Lowry
et al. (62), using BSA as a standard.
Ribonuclease Treatment
Pancreatic ribonuclease (80 yg/ml), heated at 80
for 10 min. to inactivate DNase, was added to 0.1 ml aliquots
of an incubated reaction mixture to a final concentration
of 20 yg/ml, and incubated for an additional 10 min. at
30 (54). Acid-insoluble material remaining was determined
on filter paper disks with the polymerase assay wash
procedure (see polymerase assay).
Alkaline Hydrolysis
Product, isolated by sephadex gel filtration or by
KC10^ precipitation from an incubated reaction mixture,
was suspended in 1.0 ml of 0.3 M KOH and incubated at 37
for 18 h in a stoppered test tube. After the 18 h hydrolysis,
the solution was acidified with 7 0 percent HC10^ and the
precipitate removed by centrifugation. The supernatant
was neutralized with 0.3 M KOH and the salt was removed
from the hydrolysate by charcoal adsorption (63). The
charcoal column was washed successively with water ( 3 ml)
and 0.1 M NH^OH (5 ml) and the nucleotides eluted with
5 ml of ethanol-NH^OH (ethanol-conc NH^OH-^O, 2:1:2 by
volume). The eluted fraction was lypholyzed to dryness and
resuspended in water.

28
Paper Electrophoresis
Samples to be electrophoresised were adsorbed to
paper strips (4 x 30 cm, Whatman #1) by repeatedly apply
ing and drying on one spot (5 to 10 mm dia). The paper
strips were then subjected to electrophoresis in 0.025 M
sodium citrate, pH 3.5, for 4 h at 300 v, 6-8 amps, at
25 in a Universal Electrophoresis. Cell (Buchler Instru
ments) .
Polyacrylamide Gel Electrophoresis
Polyacrylamide gels for native proteins were prepared
and run according to the method of Davis (64) using a
stacking gel and 5.2 percent polyacrylamide gels. The
gels were run at pH 8.8 and stained with aniline blue
black. The 10 percent polyacrylamide gels and denatured
enzyme protein were prepared and run according to the
method of Weber and Osborn (65). The 10 percent gels were
run at pH 7.0 in 0.1 percent SDS and stained with coomassie
brilliant blue.
Materials
DEAE-cellulose, Bio-RAD, Cellex-D (6.1 meq per g,
dry weight), was washed by the method of Peterson and Sober
(66), equilibrated in 0.05 M Tris-HCl, pH 7.6 at 25, and
stored at 4o. Sephadex G50, medium grade, Pharmacia, was

29
swollexu.a:ri£l .^jillibrated in 0.05 M Tris-HCl, pH 7.6 at 25,
and;^rfcor^di''ai(i:4^?,^Bovine serum albumin, 5X crystallized,
was..:£:rom PentexSEiochemicals. The nucleoside triphosphates
GTP, CTP, UTP -and. ATP were purchased as sodium salts from
Schwarz/Mann, including [8-l^C]ATP, [2--^C]UTP and [a-32p]
ATP. Calf thymus DNA was purchased from Schwartz/Mann, and
pancreatic ribonuclease, chromatographically pure, was
purchased from Worthington Biochemicals. Actinomycin D
was purchased..tfr:em Schwarz/Mann and cordycepin, grade C,
was purchased from Sigma. Alpha-amanitin was a gift of
Dr. T. Weiland and rifampicin was a gift from Gruppo-
Lepetit S.P.A. Research Laboratory. Zea mays L., WF9 x
Bear 38, waxy, was purchased from the Bear Hybrid Seed Co.
Reagents
Calf thymus DNA was dissolved in 0.1 x SSC at 37
ml and stored in 0.2 ml aliquots at -17 (67). An aliquot
was thawed for use before each assay. Denatured DNA was
prepared by the dilution of a freshly thawed aliquot of
DNA in 0.1 x SSC (1:1 v/v) into a sealed vial, 5 min.
immersion in boiling water, followed by quick chilling^in
iced-water (average hyperchromicity 24%). All the &260
measurements in the tables and figures represent the
absorbancy before denaturation.
Stock solutions of 1.0 M Tris-HCl, pH 8.0 or pH 7.6
at 25, 1.0 M MgC^, 14.7 M 2-mercaptoethanol were used to
make the following buffers: Buffer H, 0.25 M sucrose,

30
0.10 M Tris-HCl, pH 8.0, 1.0 mM MgC^/ and 50 mM 2-mercap-
toethanol; Buffer E, 0.05 M Tris-HCl, pH 7.6, 10 mM
2-mercaptoethanol and 0.20 M (NH^^SO^; Buffer R, 0.05 M
Tris-HCl, pH 7.6, 10 mM 2-mercaptoethanol. Buffers were
made immediately before use, using warm glass~distilled
water to minimize gas bubbles in the column eluates passing
through the spectrophotometer flowcell.
Saturated ammonium sulfate was kept at 4 with
crystals visible in the bottom of the bottle. Before use
in protein precipitations, concentrated NH^OH was added
drop-wise until a 1:20 dilution of the (NH^^SO^ was pH 8
at 25 (67). Sodium chloride-sodium citrate buffer (SSC)
1 X, was 0.15 M NaCl, 0.015 M sodium citrate, pH 7.3 at
25 (68) .

RESULTS
RNA Polymerase Purification
Utilizing the RNA polymerase .purification procedure
described in METHODS, RNA polymerase was purified 200-fold
over the 200,000 x g supernatant activity, or 1,000-fold
over that, present in the tissue homogenate. The results
of several RNA polymerase purifications are summarized in
Table 1. The steps in purification correspond to those
in Figure 1. In 10 h, 2 mg of RNA polymerase were purified
from a 90 g packet of maize roots and shoots, with a re
covery from 55 percent to 69 percent of the initial AMP
incorporating activity present in the 200,000 x g super
natant. Variability in protein and activity of the 200,000
x g supernatant probably resulted from variability in
homogenization with the Waring blender. Activity detected
in the 50 percent (NH^^SO^ precipitate was variable
(Table 1), resulting from the presence of nucleases and
variable amounts of salt in the resuspended protein. After
salt equilibration on sephadex G50, assays of polymerase
activity were less variable.
Both gradient and batch elution of DEAE-cellulose
resulted in highly active RNA polymerase. Gradient-eluted
RNA polymerase had a higher peak specific activity (104 nmoles
31

32
TABLE 1
STEPS IN RNA POLYMERASE PURIFICATION
A standard reaction mixture contained 0.37 A_,n DNA, 1.0 ymole
Zb U
MgC2/ and enzyme ,as follows: 96 yg 200,000 x g supernatant,
500 yg 50% ASP, 250 yg sephadex G50, or 3 to 5 yg DEAE-
cellulose fraction.
Step
Protein
Specific
Activity
Total
Activity
Yield
(mg)
(nmoles AMP/mg)(units)
(%)
200,000 x g Supernatant2
507+100
0.570.12
29090
100
50% ASP2
207 50
0.72+0.3
19560
6730
Sephadex G5Qa
250 50
0.700.07
17520
6010
DEAE-cellulo s e
Q
Gradient elution
2.0
66.0
132
55
ci
Batch elution
5.0
40.0
200
69
*
3.
Average of 3 preparations.
^Flowthrough contained less than
5% of added
RNA polymerase
activity.
r ...
'"'Gradient elution, peak tube specific activity, 10:4 nmoles
AMP/mg; protein, 0.2 mg/ml.
^Batch elution (0.4 M (NH
:,)oSO),
peak tube specif ic activity,
90 nmoles AMP/mg protein 0.5 mg/ml.

33
AMP/mg}', however, it was more dilute (0.2 mg/ml) and its
preparation more time consuming (6 h). Batch eluted RNA
polymerase had a lower peak specific activity (90 nmoles AMP/
mg), but it was more concentrated (0.5 mg/ml), more rapidly
eluted (2 h) and the net recovery was better than with the
gradient procedure.
The profile of &2QQ materaal during gradient elution
of the DEAE-cellulose column indicated most of the material
failed to bind to the resin equilibrated with 0.2 M (NH^)
SO^(Figure 3). However, all of the RNA polymerase activity
was bound to the resin. Of the two partially resolved
peaks of A28Q aksorfrani3 material eluted from the resin by
the (NH^^SG,, gradient, shown in more detail in Figure 4,
only the second peak exhibited RNA polymerase activity.
Although the RNA polymerase activity detected with dena
tured DNA was twice that detected with native DNA, the
ratio of specific activities with native and denatured
DNA was constant among the eluted fractions of the second
peak. Attempts to enhance purification by equilibration
of the resin with higher than 0.2 M (NH^^SO^ to reduce
binding of the protein in the first peak (Figure 4) were
unsuccessful. If the salt concentration was increased to
0.21 M, 15 percent of the enzyme activity appeared in the
column flowthrough.

ABSORBANCE AT 280nm
Figure 3. DEAE-CELLULOSE ELUTION PROFILE
AMMONIUM SULFAT

ABSORBANCE AT 280nm
Figure 4. RNA POLYMERASE ACTIVITY IN
DEAE-CELLULOSE FRACTIONS
Assayed in a standard reaction mixture
containing 0.5 nmoles MnCl9, 0.037 A260
DNA; either denatured (solid triangles)
or native (open triangles) and *2t to 4 yg
enzyme.
AMP INCORPORATED nmoles

36
Polyacrylamide Gel Electrophoresis
Antaliquot of the proteins showing the highest RNA
polymerate specific activity (eluted from DEAE-cellulose
at 235 ml in Figure 4) was examined by electrophoresis on
5.2 percent polyacrylamide gel. Several components were
visible, with more than 10 bands staining with aniline
blue black (Figure 5). All the protein staining material
migrated into the gel, toward the anode at pH 8.8. There
fore, the proteins are acidic and none were excluded by
the 5.2 percent polyacrylamide gel. The slowest migrating
and darkest staining band, however, may represent an a gregate of some of the more rapidly moving components. The
intensity of this major band was approximately equal to
that of the BSA standard (20 ug) and, therefore, may account
for 50 percent of the added protein. If an aliquot of the
same protein preparation was denatured with 0.1 M 2-mercap-
toethanol and 0.1 percent SDS, then subjected to electro
phoresis on 10 percent polyacrylamide-0.1 percent SDS gels,
the number of visible bands was reduced to 5 (Figure 6).
Two distinct bands, migrating 13 and 14 mm into the gel, a
faint band at 19 mm ^nd a still fainter pair of thin bands
at 21 and 23 mm were characteristic of all the purified
maize RNA polymerase preparations. The decrease in detect
able bands on SDSpolyacrylamide as compared to the 5.2 per
cent polyacrylamide gels may reflect disaggregation of the
denatured proteins. Alternatively, the presence of more

37
<-)
A H B
Figure 5. POLYACRYLAMIDE GEL ELECTROPHORESIS OF
NATIVE RNA POLYMERASE
Samples were run on 5.2% polyacrylamide gels at pH 8.8
(see METHODS). Gel A contained 40 yg peak RNA poly
merase (eluted at 235 ml, Figure 4). Gel B contained
20 yg BSA.

38
c)
Figure 6. POLYACRYLAMIDE GEL ELECTROPHORESIS OF
DENATURED RNA POLYMERASE
Samples were denatured in 0.1% SDS, 0.14 M 2-mercapto-
ethanol at 37 and run on 10% polyacrylamide gels
containing 0.1% SDS at pH 7.0 (see METHODS). Gel A
contained 20 yg peak RNA polymerase (eluted at 235 ml,
Figure 4). Gel B contained 6.2 yg denatured BSA.
Gel C contained denaturing buffer only.

39
bands on the gel run with native protein may reflect greater
sensitivity of band detection since the sample analyzed con
tained 'twice as much protein and the gel was stained with
aniline blue black rather than coomassie brilliant blue.
Assuming that the distance migrated by the denatured protein
components in SDS-polyacrylamide gels was a function of
their molecular weights, then the pair of bands migrating
21 and 23 mm (equivalent to the migration of BSA) were
.about 65,000 to 75,000 d. Therefore, the other bands
characteristic of the RNA polymerase preparations corre
sponded to polypeptides of higher molecular weight.
Enzyme Stability
Storage temperature and freeze-thaw
Maize RNA polymerase collected in glass vials and
immediately stored in liquid nitrogen lost no activity
after 3 months. The enzyme was stable for 19 days at -16,
but lost all activity at -17 (Table 2, part A). The
addition of glycerol to 20 percent by volume to fresh
enzyme preparations did not stabilize the polymerase
enough to make storage at -17 practical. However, in 20
percent glycerol the enzyme did retain 50 percent activity
for 17 h at 4 (Table 2, part B). The enzyme stored in
liquid nitrogen was stable to repeated freeze-thaw. The
loss of activity observed may reflect decay accumulated
during the time the enzyme was held on ice preceding assay
(Table 2, part C).

40
TABLE 2
STORAGE AND FREEZE-THAW LABILITY OF RNA POLYMERASE
After the indicated treatment aliquots of one enzyme
preparation were assayed for RNA polymerase activity in a
standard reaction mixture containing 1.0 pmole MgClj,
0.37 A.26Q native DNA and 18 yg enzyme. 100% incorporation
was equivalent to 172 pmoles AMP incorporated per reaction.
% Incorporated
A) Temperature (19 days)
-76 (Reveo freezer) 98
-17 (Freezer) 1
B) Glycerol Storage5 (17 h)
-196 (Liquid nitrogen) 96
-17 (Freezer) 78
4 (Refrigerator) 48
b
C) Freeze-thaw^
One 100
Two 100
Three 86
Four 82
Five 79
Storage on Ice (5 h)C 80
aGlycerol was added to 20% by volume to the enzyme preparation
just prior to storage.
A. vial of enzyme frozen in liquid nitrogen was thawed at 37
and held on ice prior to assay. After assay it was refrozen
in liquid nitrogen. This sequence was repeated as indicated.
CEnzyme frozen and thawed once prior to 5 h storage on ice.

41
Salt precipitation
-.w jtotasr.a.t^Tempt to concentrate the RNA polymerase eluted
sfrccra- DEX2-:eelUiXose, solid (NH^^SO^ was added to 8 0 per
cent saturation to the pooled fractions. Only 10 to 15 per
cent of the eluted activity was recovered in the precipitated
protein. To facilitate precipitation, calf thymus DNA was
added in varying amounts to the pooled fractions just before
salt addition. Addition of DNA resulted in an increase in
precipitated activity, roughly porportional to the amount
of DNA added (Table 3). Low recovery of polymerase in
precipitates from a solution with high DNA levels (300 pg/
ml) suggested ..formation of a DNA-protein complex soluble in
high salt. Neither polymerase activity in the supernatants
nor protein in the precipitates was determined, therefore
inactivation of polymerase and specific activity were not
assayed per se.
Summary
DNA-dependent RNA polymerase was purified 1,000-fold
from an homogenate of maize shoots and roots. Following
DEAE-cellulose chromotography the enzyme specific activity
was greater than 100 nmoles AMP incorporated/mg enzyme at
20 min. The recovery was greater than 50 percent of the
activity detected in the high speed supernatant. Poly
acrylamide gel electrophoresis of both native and SDS-
denatured proteins indicated the RNA polymerase preparation
still contained several polypeptides. Nevertheless the

42
TABLE 3
SALT PRECIPITATION OF RNA POLYMERASE
Each sample contained 0.75 mg protein before 80% saturation
with solid, powdered (NH^^SO^. Just before addition of
salt, DNA was added as indicated, then salt was slowly add
ed while the mixture was stirred on ice for 15 min. The
precipitate was collected by centrifugation (10,000 x g for
10 min.), resuspended and desalted in Buffer R on sephadex
G25. Samples were assayed in a standard reaction mixture
with 1.0 ymole MgCl2 and 0.37 A2gQ DNA.
DNA Added (yg/ml)
% Activity Recovered3
None
13
2.4
20
6.0
30
12.0
38
30.0
46
300.0b
10
a
Control incorporated 185 pmoles AMP.
Very little precipitate obtained.
I

43
denatured protein exhibited those bands characteristic of
eukaryotic RHA polymerases. The enzyme wat stable after
'freezing and-storage at -7 6. Precipitation of the purified
enzyme with (NH^)2S04 was facilitated by DNA. Further
resolution of the proteins in the RNA polymerase preparation
was thwarted by the instability of the enzyme at 4 and by
the lack of recovery of active enzyme following salt
precipitation.
RNA Synthesis
Assay Requirements
Enzyme purified by the procedure described in METHODS
exhibited the expected requirements for RNA synthesis.
DNA, metal and all four ribonucleosidetriphosphates were
required for incorporation of labeled AMP into acid-insoluble
v
material (Table 4). Denatured DNA was utilized as well as
native calf thymus DNA, and maize DNA (not shown here) sup-
2+
ported comparable activity. Mn satisfied the metal re-
2+ 2+
quirement as well as Mg in fact, better than Mg when
denatured DNA was provided as the nucleic acid component.
*
Incorporation of AMP in the absence of NTPs was significant,
especially when assayed in the presence of denatured DNA.
All of the radioactivity detected in acid-insoluble material
on filter paper disks was dependent upon the addition of
enzyme protein.

44
TABLE 4
ASSAY REQUIREMENTS FOR MAIZE RNA POLYMERASE
A standard reaction mixture contained 0.37 A,n DNA, 1.0 ymole
A DU
MgCi^ or 0.5 ymoles MnCl^ and 5 yg RNA polymerase.
System
AMP Incorporated3 (pmoles)
Native DNA Denatured DNA
Complete 235 327
Complete less DNA 0 1
2+
Complete less Mg 0 1
Complete less Mg^+, plus Mn^+ 300 551
Complete less NTPs 10 58
Complete less enzyme 0 0
lTotal radioactive AMP incorporated per reaction mixture.

45
DNA
,iv 2+
^fte';'.c'orpe'te system with Mg as the metal cofactor,
the-'B'NA' requirement- was satisfied with either native or
heat-denatured DNA (Table 4). The reaction was saturated
with denatured DNA in excess of 0.4 A260//m'*"' whereas with
native DNA more than a 5-fold higher concentration (1.8 A2g(/
ml) was required to reach the same level of activity (Fig
ure 7). If equal amounts of native and denatured DNA were
combined and /assayed in the same reaction mixture, the
activity measured could be accounted for by the denatured
DNA alone* Thus, the denatured DNA appeared to out-compete
the native DNA ;,:for the RNA polymerase. However, at
saturating concentrations native and denatured DNA supported
equivalent AMP incorporation.
If MnCl2 was substituted for MgCl2 in the reaction
mixture, there was almost a 2-fold increase in RNA polymerase
activity with denatured DNA (Table 4). In the presence of
2+
Mn the activity with denatured DNA was twice that with
native DNA over a range of DNA concentrations (Figure 8).
Although the RNA polymerase assay was saturated at 0.4 A^g/
ml with both native and denatured DNA, AMP incorporation
after 20 min. was not equivalent. Therefore, the concen
tration dependence of the polymerase on native and denatured
2+
DNA differed when assayed with Mg but the maximum level
2+
of activity'was the same. In contrast with Mn the con
centration dependence with native and denatured DNA was

AMP INCORPORATED pmoies
Figure 7. DNA TITRATIONS WITH MAGNESIUM AS COFACTOR
Assayed in a standard reaction mixture containing 1.0 ymole MgCl2
4 yg RNA polymerase and DNA as indicated; native (solid circles) ,
denatured (solid triangles) and native-denatured mixture (open circles) .

AMP INCORPORATED pmoles
Figure 8. DNA TITRATIONS WITH MANGANESE AS COFACTOR
Assayed in a standard reaction mixture containing 0.5 pmoles
MnCl2/ 4 pg RNA polymerase and DNA as indicated; native (solid
circles) or denatured (solid triangles).

48
similar but the maximum level of AMP incorporation differed.
Metal
For maize RNA polymerase activity there was an
absolute 'requirement for a metal ion cofactor satisfied
2+ 2 +
with, either Mg or Mn (Table 4) There was a broad
2-§-
Mg optimum concentration from 10 'to 14 mM, centered at
12 mM, and independent of the nativity of the DNA (Figure 9).
Routinely, however, 10 to 25 percent more activity was
observed with denatured DNA than with native DNA. The
2+
maximum AMP incorporation occurred with 5 mM Mn either
with native or denatured DNA (Figure 10). With denatured
DNA, the RNA polymerase activity was twice that with native
2+
DNA at all Mn levels. As is characteristic of polymerases,
2+
the Mn optimum concentration was sharper than that of
,, 2+
Mg
Substrate
With native DNA, 96 percent of the AMP incorporation
required the presence of the NTPs (Table 4). The RNA poly
merase reaction was saturated with the pooled NTPs above
1.0 mM for each of the three nucleoside triphosphates;
GTP, CTP and UTP and no nucleotide inhibition was detected
with a 5-fold excess (Figure 11). The standard reaction
was 2.5 mM with each NTP; thus the assay of polymerase was
not limited with respect to unlabeled nucleosidetriphosphates
The RNA polymerase reaction mixture was saturated

AMP INCORPORATED pmoles
49
Figure 9. MAGNESIUM TITRATION WITH
NATIVE AND DENATURED DNA
Assayed in a standard reaction mixture
containing 0.37 A?60 DNA; native (solid
circles) or denatured (solid triangles),
6.4 yg RNA polymerase and MgC^ as
indicated.

AMP INCORPORATED pmoles
50
Figure 10. MANGANESE TITRATION WITH
NATIVE AND DENATURED DNA
Assayed in a standard reaction mixture
containing 0.37 &260 DNA? native (solid
circles) or denatured (solid triangles),
6.4 \ig RNA polymerase and MnC^ as indicated.

AMP INCORPORATED pmoles
51
Figure 11. NTP TITRATION OF RNA POLYMERASE
Assayed in a standard reaction mixture con
taining 1.0 uniles MgC^/ 0.37 nat:*-ve
DNA, 6.4 yg RNA polymerase and pooled NTPs
(GTP, CTP, UTP) as indicated.

52
with ATP above 1.0 mM (Figure 12). A Lineweaver-Burke
. -4
reciprocal plot resulted in a of 1.25 x 10 M ATP
(Figure 13). The Km was the same with native and dena
tured DNA, although the V with denatured DNA was twice
that with native DNA. The standard ATP concentration for
the RNA polymerase reaction mixture was 1.0 mM, since it
resulted in the highest ATP specific activity while main
taining the maximum rate of RNA synthesis. The rate of
AMP incorporation decreased when the ATP concentration was
below saturation. If the ATP concentration was decreased
to 0.5 mM there was 86 percent of the maximum rate and if
it was decreased to 0.25 mM there was 68 percent of the
maximum rate. The pmoles of AMP incorporated per reaction
was independent of the [8-l4C]ATP specific activity (see
APPENDIX A.).
Salt
As with the partially purified enzyme, the more
highly resolved preparations exhibited a dependency on
added (NH^^SO^ for RNA polymerase activity, especially
in the presence of native DNA (Figure 14). The maximum
level of activity occurred between 60 and 90 mM (NH4)2S04
with native DNA, and up to 100 mM with denatured DNA.
Because the RNA polymerase fraction contained 0.4 M
(NH^)2S0^, the titrations at concentrations below 40 mM
were not performed.

53
Figure 12. ATP TITRATION OF RNA POLYMERASE
Assayed in a standard reaction mixture containing
1.0 ymole MgC^/ 0.37 nati-ve NA, 5 yg RNA
polymerase andzATP as indicated.

54
0 4 8
I
ATP
Figure 13. LINEWEAVER-BURKE PLOTS OF ATP
TITRATIONS
Assayed- in a standard reaction mixture con
taining 0.5 limles MnCl2, 0.37 A2gg DNA, either
native (solid circles) or denatured (solid
triangles), and 4 yg RNA polymerase. The ATP
concentration was mM with the K at 0.125 mM.
The was 100 moles AMP/mg/2U min. for de-
nature§XDNA and 50 nmoles AMP/mg/20 min. for
native DNA.

AMP INCORPORATED pmelsG
55
t
... t I
ICO
AMMONIUM SULFATE mM
Figure 14. AMMONIUM SULFATE TITRATION OF
RNA POLYMERASE
Assayed in standard reaction mixture con
taining 1.0 ymole MgCl^> -37 &260 DNA'
either native (solid circles) or denatured
(solid triangles), 6.4 yg RNA polymerase and
(NH^^SO^ as indicated.
200

56
Enzyme
If enzyme protein was not added there was no AMP
incorporation in the standard RNA polymerase reaction
mixture (Table 4). The total AMP incorporated in 20 min. was
the function of the enzyme protein added, up to 6 yg pro
tein per standard reaction mixture .(Figure 15). In the
presence of the NTPs, the amount of AMP incorporated with
denatured DNA.was twice that incorporated with native DNA,
at all enzyme concentrations.
Rate of AMP Incorporation
The amount of AMP incorporated in a standard RNA
polymerase reaction increased as a function of time for
more than 90 min. (Figure 16). The rate of AMP incorpora
tion decreased continuously during the initial 20 min. of
incubation and then became constant. The initial rate
(R^) and the late rate (R£) were arbitrarily defined as
that occurring up to 20 min., and that occurring after
20 min. What appeared to be two different reactions
(initial and late) were not resolved by increasing the
incubation temperature from 30 to 37.
Nearest Neighbor Frequency
Product synthesized on denatured calf DNA with
[a32p]ATP after alkaline hydrolysis, yielded four labeled
nucleotides (Table 5) Since the product was acid-insoluble
and excluded from sephadex G25, indicating an oligomer,
the nearest neighbor frequency data indicated the product

AMP INCORPORATED pmotes
Figure 15. RNA POLYMERASE ACTIVITY AS A
FUNCTION OF ENZYME CONCENTRATION
Assayed in a standard reaction mixture con
taining 0.5 pinoles MnC^? 0.37 A^gg DNA,
either native (solid circles) or"' denatured
(solid triangles), and enzyme protein as
indicated.
~5 *0

AMP INCORPORATED pmoles
58
0 50 100
TIME min
Figure 16. RATE OF AMP INCORPORATION AS A
FUNCTION OF TIME
Assayed in a standard reaction mixture (7X) each
containing 1.0 ymole MgC^/ 0.37 A260 na'^:'-ve
and 18 ug RNA polymerase. The reactions were
incubated at either 30 (solid circles) or 37
(solid triangles) and 0.1 ml aliquots removed at
the times indicated.

59
TABLE 5
NEAREST NEIGHBOR FREQUENCY ANALYSIS OF PRODUCT
SYNTHESIZED WITH DENATURED DNA
A standard reaction mixture containing 0.37 denatured
DNA, 0.10 nmoles [a-32p]ATP (specific activity 30 mCi/mmole) ,
0.4 ymoles MnC^ and 10 ng enzyme was incubated at 30 for
60 min. 1% SDS was added to the reaction, it was passed
over sephadex G25, the material in the excluded volume was
hydrolyzed in 0.3 M KOH for 17 h at 37, adsorbed to acti
vated charcoal and the eluted nucleotides lyophilized, re
suspended in H2O and resolved by paper electrophoresis.
(see METHODS).
CPM
Added Recovered9,
CpA ApA GpA UpA
55,000 27,600
0.25 0.44 0.18 0.13
aSome of the sample was lost during lyophilization.

60
was RNA. The relatively high ApA frequency derived from
product synthesized with denatured DNA suggested the
presence of poly(A) regions in the product.
Summary
The maize RNA polymerase required added DNA, the
four nucleosidetriphosphates; ATP, GTP, CTP and UTP, and
2+ 2+
a metal, either Mg or Mn for activity. The maximum
activities with native and denatured DNA were equal when
2+
Mg was a cofactor. The maximum activity with denatured
. 2+
DNA was twice that with native DNA if Mn was the metal
cofactor. Enzyme activity was salt-dependent and pro
portional to the amount of enzyme protein added. The
substrate reached saturation above 1.0 mM and the reaction
continued for at least 90 min. The product with denatured
DNA had a high ApA nearest neighbor frequency.
Polyadenylic Acid Synthesis
The RNA synthesis data indicated that an additional
activity might be present in the RNA polymerase reaction.
These indications were: 1) With denatured DNA there was
significant incorporation of AMP in the absence of the NTPs.
2) There was a doubling in activity with denatured DNA
2+ 2+
when Mn replaced Mg but with native DNA there was little
change. 3) There appeared to be a biphasic rate of AMP

61
incorporation, one rate at early times and another at late.
TheserreBuis'suggested poly(A) might be synthesized early
in a ire-actioxi mixture containing denatured DNA.
Assay Requirements
The standard reaction mixture for poly(A) synthesis
was the same as that for the RNA polymerase assay (see
METHODS) except the NTPs were omitted.
DNA
2+
With Mg as cofactor, AMP was incorporated with
denatured DNA in the absence of the NTPs (Table 4). With
denatured DNA the reaction was saturated at 0.2 A2gQ/ml
(Figure 17), or about half that required to saturate the
reaction in the presence of NTPs (Figure 7). The maximum
AMP incorporated with native DNA did not exceed 5 percent
of the AMP incorporated with denatured DNA.
2+
With Mn as cofactor, there was a 2-fold increase
in AMP incorporation with denatured DNA as compared with
2+
Mg but the denatured DNA still saturated the reaction
mixture at 0.2 (Figure 18). Native DNA supported
a much lower rate of incorporation ranging from 8 percent
to 15 percent of the activity with denatured DNA.
Substrate
AMP incorporation in the absence of the NTPs was
first detected with denatured DNA (Table 4). In the
2+ 2+
presence of Mn rather than Mg only half of the AMP

AMP INCORPORATED pmoles
Figure 17. DNA TITRATIONS WITH MAGNESIUM IN THE ABSENCE OF NTPs
Assayed in a standard reaction mixture containing 1.0 ymole MgCl2, 4yg RNA polymerase
and DNA, either native (solid circles) or denatured (solid triangles), as indicated.
fO

J200r
O
E
Ol
DMA A26o/ml
Figure 18. DNA TITRATIONS WITH MANGANESE IN THE ABSENCE OF NTPs
Assayed in a standard reaction mixture containing 0.5 ymole MnCl^
4 yg RNA polymerase and DNA, either native (solid circles) or de
natured (solid triangles), as indicated.
cn
co

64
incorporation on denatured DNA was dependent upon the
other NTPs (Table 6). In contrast essentially all the
incorporation of radioactive UMP required the other NTPs.
These results were consistent with the simultaneous syn
thesis of polyadenylic acid and RNA. Furthermore, they
suggested that homopolymer synthesis might be limited to
poly(A).
A titration of the NTP requirement indicated that
at low NTP concentrations (0.025 mM) AMP incorporation
was inhibited, whereas at higher NTP concentrations AMP
incorporation was stimulated (Figure 19). Except for the
activity observed with no NTPs, the stimulatory portion
of this titration resembled that with native DNA (Figure 11)
Addition of GTP, CTP or UTP individually at 3 mM resulted
in 80 percent inhibition of AMP incorporation (Table 7).
The addition of UTP at 0.3 mM (12% of the standard assay
concentration) inhibited AMP incorporation to the same
degree. The inhibitory effect of low NTP concentrations
was observed upon addition of the NTPs before or after
addition of the enzyme and incubation of the reaction mix
ture (Table 8). If 0.025 mM NTPs (1% standard NTP con
centration) were added just after enzyme addition, after
1 min. incubation or after 5 min. incubation at 30, in
all cases it resulted in inhibition of AMP incorporation
(Table 8). The total AMP incorporated per reaction
increased the longer the addition of NTPs was delayed,

65
TABLE 6
AMP AND UMP INCORPORATION BY RNA POLYMERASE
A standard reaction mixture containing 0.37 A2gQ denatured
DNA, 0.4 ymoles MnCl2/ either 0.10 ymoles labeled ATP or
0.05 ymoles labeled UTP and 4 yg enzyme.
Labeled Nucleotide Incorporated
System (pmoles)
14 14
C-AMP C-UMP
Complete
381
Complete plus NTPs
913
22
458C
aTotal radioactive nucleotide incorporated per reaction
mixture.
NTPs
contained
0.25
ymoles
each:
GTP,
CTP
and
UTP .
NTPs
contained
0.25
ymoles
each:
GTP,
CTP
and
ATP.

6 6
NTPs mM
Figure 19. NTP TITRATION WITH DENATUREDDNA
Assayed in a standard reaction mixture containing
0.037 denatured DNA, 0.5 timles MnCl- 5 yg
RNA polymerase and .pooled NTPs (GTP, CTP, and UTP)
as indicated (mM each).

67
TABLE 7
INHIBITION OF POLYADENYLIC ACID SYNTHESIS
BY NUCLEOTIDES
RNA polymerase activity measured in a standard reaction
TT!'ixt.ure containing 0.037 A260 denatured UNA, 0.05 ymoles
.MnC.12 / 5 yg enzyme, and nucleotides as indicated.
Additional Nucleotides AMP Incorporated3 (pmoles)
GTP, CTP, UTP (0.25 ymoles each) 619
None 210
UTP (0.3 ymoles) 40
GTP (0.3 ymoles) 31
CTP (0.3 ymoles) 43
UTP (0.03 ymoles) 50
a
Total pmoles AMP incorporated per reaction mixture.

68
TABLE 8
EFFECT OF DELAYED ADDITION OF NTPs ON
POLYADENYLIC ACID SYNTHESIS
A standard reaction mixture containing 0.5 ymoles MnC1^>
0.037 &260 denatured DNA, 5 yg enzyme and NTPs as indicated
were \ised. Delayed addition of the NTPs was at times in
dicated during a standard 20 min. incubation. NTPs con
tained GTP, CTP and UTP.
NTPs Added
AMP Incorporated5
pmoles %
None 160 40
0.25 yiQoles each, 0 min. 400 100
0.0025 pmoles each,
0 min. 52 13
1 min. 60 15
5 min. 88 22
aTotal pmoles incorporated per reaction mixture.

69
consistent with the synthesis of poly(A) before NTP addition.
Enzyme
Enzyme was required for AMP incorporation and
increased as a linear function of the enzyme protein present
(Figure 20). Replacement of denatured DNA with native DNA
caused greater than a 90 percent decrease in AMP incorpora
tion. Therefore, the synthesis of poly(A) was directly
proportional to the enzyme level and the enzyme preferred
denatured DNA.
Product Characterization
The acid-insoluble product synthesized with native
2+ 2+
or denatured DNA with either' Mg or Mn as cofactor
remained insoluble after heating at 100 in 1 percent SDS
for 5 min. (Table 9). As anticipated, if poly (A) longer
than 12 nucleotides was accumulated in the reaction with
denatured DNA, a more acid-insoluble product was synthesized
and it too remained acid-insoluble after treatment.
Nearest neighbor frequency analysis of products
3 2
synthesized from [a- P]ATP in the.absence of the NTPs
resulted in a 0.90 ApA frequency (Table 10), indicating
poly(A) synthesis. Incorporation into CpA, GpA and UpA
accounted for 10 percent of the label incorporated.
The average chain length of the product was estimated
by the ratio of AMP to adenosine after alkaline hydrolysis.
The chromatographed AMP fraction contained 2,200 cpm,

AMP INCORPORATED pmoles
70
PROTEIN pg
Figure 20. POLYADENYLIC ACID SYNTHESIS AS A
FUNCTION OF ENZYME CONCENTRATION
Assayed in a standard reaction mixture containing
0.5 ymole MnCl-/ 0.37 DNA, either native
(solid circlesf or denatured (solid triangles),
and enzyme protein as indicated.

71
TABLE 9
EFFECT OF HEATING AND SDS ON
RNA POLYMERASE PRODUCT
Standard reaction mixture with 0.37 A2gQ DNA, and either
1.0 ymoles MgCl2 or 0.4 ymoles MnCl2 as indicated. Heating
was for 5 min. at 100. SDS was 1% where indicated. Each
assay contained 6.5 yg enzyme.
Components in Reaction Mixture
AMP Incorporated
(pmoles)*
Template
Metal
Control
Heated
Heated in SDS
Native
Mg
660
610
625
Native
Mn
627
632
630
Denatured
Mg
805
800
840
Denatured
Mn
1160
1160
1190
Total radioactive nucleotide incorporated per reaction
mixture.

72
TABLE 10
NEAREST NEIGHBOR FREQUENCY ANALYSIS OF
POLYADENYLIC ACID PRODUCT
A standard reaction mixture containing 0.37 A2gQ denatured
DNA, 0.10 ymoles [a-32p]ATP (specific activity 30 mCi/mmole) ,
0.4 ymoles MnCl2 and 10 yg enzyme was incubated at 30 for
60 min. 1% SDS was added to the reaction, it was passed
over sephadex G25, material in the excluded volume was
hydrolyzed in 0.3 M KOH for 18 h at 37, adsorbed to acti
vated charcoal and the eluted nucleotides lyophilized, re
suspended in H20 and resolved by paper electrophoresis (see
METHODS),
CPM
Added
Recovered
a
CpA ApA GpA UpA
11,000 4,500 0.01 0.90 0.04 0.05
iSome of the sample was lost during lyophilization.

TABLE 11
AVERAGE CHAIN LENGTH OF POLYADENYLIC ACID
A standard reaction mixture was used containing 0.037 A26Q
denatured DNA, 0.5 ymoles MnCl2/ no NTPs and 12 yg RNA
polymerase. The reaction was incubated for 60 min. at 30.
Yeast RNA (1 mg) was added and the product precipitated by
the addition of 0.3 ml of cold 1.0 M HC10^ containing 5 mM
sodium pyrophosphate. The ppt. was washed 4X with 1 ml of
cold 0.5 M HC10^ and then alkaline hydrolyzed and
electrophoresised (see METHODS).
Compound CPM
AMP
2,200
Adenosine
<22

74
whereas the radioactivity detected in the adenosine fraction
did not exceed background (Table 11). The sensitivity of
the assay for adenosine was 22 cpm; therefore the lower
estimate of the average chain length for poly(A) was 2,200/
22, or greater than 100 nucleotides per chain after 60 min.
at 30.
Summary
Maize RNA polymerase synthesized polyadenylic acid in
the presence of denatured DNA, ATP and metal, preferably
2+
Mn There was greater than a 17-fold preference for
ATP over UTP as substrate for homopolymer synthesis. Low
levels of the NTPs as a mixture or added individually in
hibited poly(A) synthesis, independent of the time of
addition. Poly(A) synthesis was directly proportional to
the amount of enzyme protein added. The product remained
acid-insoluble after boiling with.. SDS. The average chain
length of the product accumulated after 60 min. incubation
was greater than 100 nucleotides and the ApA frequency was
0.90.
Comparison of RNA and Polyadenylic
Acid Synthesis
The two AMP incorporating activities measuring RNA
and poly(A) synthesis were apparently catalyzed by the same
enzyme; RNA polymerase II (69). The evidence for this

75
conclusion was indirect, since the enzyme preparation was not
homogeneous. The data presented thus far in support of one
enzyme catalyzing both syntheses includes: 1) Both activ
ities were eluted simultaneously from DEAE-cellulose by
an (NH^^SO^ gradient with a constant RNA and poly (A) activity
ratio in the active fractions. 2) Both activities required
DNA for AMP incorporation. 3) Both activities had the
2+ 2+
same Mg and Mn cofactor optima. 4) The for ATP was
the same with native and denatured DNA. Additional com
parisons of RNA and poly(A) synthesis were made to further
associate the accumulation of both products with one
enzyme. These included inhibitor activities, nuclease
sensitivity of products as a function of time and template
specificity.
Effect of Inhibitors
Three types of inhibitors of RNA polymerase were
investigated, those that bind specifically to the enzyme
protein such as a-amanitin and rifampicin; those that
bind to the template such as actinomycin D; and those
that compete with the substrate such as cordycepin. The
inhibitors were tested both in the presence and absence
of the NTPs, therefore measuring the sensitivity of both
RNA and poly(A) syntheses.
Low concentrations of a-amanitin inhibited both RNA
and poly(A) activity equally (Table 12). A titration of
RNA polymerase activity with increasing levels of a-amanitin

76
TABLE 12
INHIBITOR SENSITIVITY OF MAIZE RNA POLYMERASE
A standard reaction mixture contained 0.037 A
DNA, 0.5 ymoles MnCl2, 0.05 ymoles ATP, 5 yg
hibitors as indicated added at zero times.
2gg denatured
enzyme and in-
Inhibitor Concentration
Percent
of Control
Plus NTPsa
Less NTPs^
a-amanitin 0.1 yM
6
7
1.0 yM
1
2
Actinomycin D 7.0 yM
14
128
70.0 yM
1
87
Cordycepin 0.13 mM
100
101
0.26 mM
-
100
Rifampicin 50.0 yg/ml
100
110
a336 pmoles AMP incorporated equaled
100%.
^138 pmoles AMP incorporated equaled
100%.

77
_ g
indicated 50 percent inhibition at 1 x 10 M (Figure 21).
Since a-amanitin is specific for the type II eukaryotic
KNA polymerase (32), this inhibition of activity indi
cated that the maize enzyme was a type II RNA polymerase
and that it catalyzed both RNA and.poly(A) synthesis (69).
Actinomycin D inhibited AMP incorporation in the
presence of the NTPs (Table 12). However, in the absence
of the NTPs, AMP incorporation was stimulated. Even at
very high actinomycin D concentrations (70 yM), there was
only a slight inhibition of poly(A) synthesis. Therefore,
the sensitivity of the RNA polymerase to actinomycin D was
dependent upon the presence of the NTPs. In the presence
of the NTPs, AMP incorporation was inhibited 50 percent
at 2 yM actinomycin D and was inhibited 95 percent at 50 yM
(Figure 22) .
The inhibition by a-amanitin and the resistance to
cordycepin and rifampicin excluded the presence of two
potential enzyme contaminants (Table 12). The maize NTP:
exotransferase is resistant to a-amanitin and sensitive
to cordycepin while bacterial RNA polymerases are resistant
to a-amanitin and sensitive to rifampicin. Therefore the
presence of NTP: exotransferase activity from maize tis
sue and bacterial RNA polymerase activity from contaminat
ing bacteria were ruled out.
Utilizing the resistance of purine-purine
phosphodiester linkages to pancreatic ribonuclease (54),

78
Figure 21. ALPHA-AMANITIN TITRATION OF RNA POLYMERASE
Assayed in a standard reaction mixture containing 1.0 jimole
MgC^r 0.37 &260 nat^-ve 18 yg RNA polymerase and
a-amanitin as indicated. Inhibitor was added just prior to
addition of enzyme to the reaction mixture.

PERCENT ACTIVITY
79
Figure 22. ACTINOMYCIN D TITRATION OF RNA POLYMERASE
Assayed in a standard reaction mixture containing 1.0 ymole
MgCl2, 0.37 A2g0 native DNA, 18 yg RNA polymerase and
actinomycin D as indicated. Inhibitor was added just prior
to addition of enzyme to the reaction mixture.

80
we determined the amount of poly(A) accumulated with dena
tured DNA in a standard reaction mixture as a function of
time. .AMP incorporation in the absence of the NTPs was
not affected by the ribonuclease treatment (Figure 23,
panel B). In data not shown here AMP incorporation in a
standard reaction mixture containing denatured DNA and
4 yg ribonuclease showed no inhibition of incorporation,
consistent with the exclusive synthesis of poly(A). In
the presence of the NTPs, there was a decrease in acid-
insoluble AMP when the product was treated with ribonuclease
(Figure 23, panel A). However, the 10-min. product was
only 10 percent sensitive to treatment whereas the 60-min.
product was 45 percent sensitive. This result suggests
early synthesis of poly(A) and later synthesis of RNA.
Template Requirement
Both calf thymus and maize DNA supported RNA and
poly(A) synthesis (Table 13). In the absence of the NTPs,
where only poly(A) was synthesized, denatured maize and
calf thymus DNA supported equal AMP incorporation. Since
poly(A) synthesis was supported by DNA from both species,
the synthesis of poly(A) was not an artifact of the
heterologous system (calf thymus DNA and maize polymerase).
The lower AMP incorporation in the complete system
observed on denatured maize DNA as compared with denatured
calf DNA reflects the presence of native DNA in the heated
maize preparation; note the low hyperchromicity of the
maize DNA.

Assayed in a standard reaction mixture containing 0.37 A denatured DNA and 0.5 ymole
MnCl2; panel A with NTPs, panel B without NTPs, and 5 yg^RNA polymerase. At the times
indicated reaction mixtures were removed and either precipitated in 10% trichloroacetic
acid or incubated with 0.2 yg pancreatic ribonuclease (see METHODS). RNase treated samples
indicated by open circles. \ 1 y
co
i

82
TABLE 13
COMPARISON OF CALF THYMUS AND MAIZE DNA AS
TEMPLATES FOR RNA POLYMERASE
Assayed in a standard reaction mixture containing 0.5 ymoles
MnC^, 0.037 A^gg DNA anc^ ^.4 ^9 P^Y617356
DNA
£
AMP Incorporated (pmoles)
Complete Less NTPs
Native Calf Thymus
219
29
Native Maize
260
46
Denatured Calf Thymus*3
400
160
. c
Denatured Maize
300
163
aTotal pmoles incorporated
per reaction
mixture.
uHyperchromicity 26%.
cHyperchromicity 11%.

83
If RNA polymerase catalyzed two reactions, RNA
synthesis and poly(A) synthesis, and if both reactions
required a template, then no AMP incorporation on
poly(dAT), the alternating copolymer, would be expected
in the absence of UTP. As seen in Table 14, AMP incorpo
ration on poly(dAT) was observed only in the presence of UTP,
Poly(dAdT), the complementary homopolymers, were
also templates' for AMP and UMP incorporation. AMP incor
poration was stimulated 2-fold by the addition of UTP,
suggesting synthesis of poly(U) on a poly(dA) template.
However, when UMP incorporation was measured directly
14
(with C-UTP) little product accumulated unless ATP was
added to the reaction mixture, suggesting that poly(A)
synthesis was required for UMP incorporation. Incorporation
with the complementary homopolymers was sensitive to
a-amanitin indicating that the RNA polymerase was catalyzing
the ATP stimulated UMP incorporation.
Summary
RNA and poly(A) synthesis by RNA polymerase II was
confirmed by the sensitivity of both to a-amanitin. Ribo-
nuclease resistance suggested that the early product was
poly(A). Requirements for poly(A) and RNA synthesis with
several deoxyoligomers demonstrated that template was
required for both poly(A) and RNA synthesis. The
differential'sensitivity to actinomycin D indicated that

84
TABLE 14
RNA POLYMERASE ACTIVITY WITH SYNTHETIC TEMPLATES
Enzyme activity assayed in a standard reaction mixture con
taining 0=5 ymoles MgCl2, 0.037 &260 DNA' either 0.1 ymole
labeled ATP or 0.05 nmole labeled UTP and 4 yg RNA poly
merase.
Nucleic Acid Substrate Incorporation (pmoles^1
AMP UMP
Poly(dAT)
14
C-ATP
13
-
Poly(dAT)
14
C-ATP
+
UTP
1,090
-
Poly(dAdT)
14
C-ATP
104
-
Poly(dAdT)
14
C-ATP
+
UTP
211
-
Poly(dAdT)
14
C-UTP
-
4
Poly(dAdT)
14
C-UTP
+
ATP
-
40
Poly(dAdT)
14
C-UTP
+
Id
ATP (a-aman)
-
0.2
aTotal radioactive nucleotide incorporated per reaction
mixture.
^a-amanitin added to 20 yg/ml.

the polymerase utilized different sequences in the DNA
but not necessarily different DNA molecules for RNA and
poly(A) synthesis.

DISCUSSION
Enzyme Preparation
Studies of both product characterization and the
initiation of transcription required an enzyme of a much
higher specific activity than the soluble maize RNA poly
merase purified by Stout and Mans (27). In addition, the
net yield of active polymerase had to be increased and the
purification procedure made more convenient and rapid for
.effective experimental progress. These goals were achieved.
The RNA polymerase specific activity was increased 10-fold,
the net yield was more than doubled, and the time required
for each preparation was reduced from 7 days to 10 hours.
These improvements resulted from 4 major alterations in the
procedure of Stout and Mans (27), including changes in:
1. the storage of the starting material; 2. the homogeniza
tion procedure; 3. the salt equilibration of the soluble
proteins; 4. the DEAE-cellulose chromotography procedure.
Previously (27), adequate grain was germinated under
running water and/after 5 days, the seedlings were harvested
just before each enzyme preparation. In contrast, after
large scale germination and harvest (1 to 3 kg) followed
by storage of the seedlings at -76 (Reveo freezer) in
86

87
aluminum packets, each enzyme preparation required only the
removal of a weighed packet of tissue from the freezer just
before homogenization. The specific activities of the
homogenates from freshly harvested tissue and from tissue
stored at -76 were identical.
In the original procedure (27), the shoots and roots
were pulverized under liquid nitrogen and then, in small
batches, passed through a French pressure cell. This was
replaced by rapid homogenization, in one batch, in a
Waring blender. During homogenization, the enzyme was
protected from oxidation (foaming) with 50 mM 2-mercapto-
ethanol. The enzyme specific activity of the blender
treated material (Table 1) was identical to material from
the French pressure cell (27).
The removal of (NH^^SO^ from the salt-precipitated
enzyme fraction by a 4 h dialysis against Buffer R (27),
was replaced by a 20 min. gel filtration procedure. The
Sephadex G50 was equilibrated and the proteins eluted in
the excluded volume with 0.2 M (NH^)2SO^. At this salt
concentration all RNA polymerase was bound to the DEAE-
cellulose column, but only a small fraction of the total
protein was bound (illustrated by A2gQ in Figure 3).
Previously (27) RNA polymerase was eluted with a
shallow Tris-HCl gradient (500 ml, 0.05 to 1.0 M). This was
replaced by a steep (NH^)2SO^ gradient (60 ml, 0.2 to 1.0 M),
that eluted concentrated, highly active, RNA polymerase

88
(Table 1). Stout and Mans (27) had concentrated the dilute
Tris-HCl eluted enzyme by salt precipitation. Because the
(KrH^) 2^0^ eluted enzyme was concentrated, the salt precipita
tion was obviated, and the 70 to 90 percent loss of activity
such precipitation, caused, was avoided. In addition to
the removal of most of the contaminating proteins, most, if
not all, nucleic acids were removed. The A,
i/A-
of the
260' 280
material added to the column was 10, whereas the A26c/A28Q
of the eluted fractions containing polymerase was 1.5 to
2.0. Removal of nucleic acids during DEAE-cellulose
chromotography apparently decreased the stability of the
'iEEA. polymerase. The polymerase was stable to salt precipi
tation before, but not after, chromotography. Addition of
DNA, to the eluted fractions containing the polymerase,
prior to precipitation with (NH^)2SO^ improved the recovery
of polymerase activity (Table 3), supporting the premise
that the polymerase requires nucleic acid for stability.
The recovery of active enzyme free of nucleic acid
may, therefore, result from the short time between elution
from DEAE-cellulose and storage in liquid nitrogen. During
the absence of nucleic acid, the RNA polymerase complex
may either aggregate or disassociate, but in either case
it becomes inactive. This hypothesis was supported by the
polyacrylamide gel electrophoresis data (Figure 5). The
presence of one dark band and multiple smaller bands might
represent subunits which have aggregated and/or disassociated
from the polymerase complex.

89
Strain et ^1. (47) reported the partial resolution
of two type II maize RNA polymerases during DEAE-cellulose
chromotography; one which preferred native DNA and one
which preferred denatured DNA. The conditions of purifi
cation resulted in very low specific activities, perhaps
reflecting an advanced state of polymerase degeneration.
The possible degeneration of RNA polymerase is supported
by the data of Chambn (39) with the detection of RNA poly
merase protein subunits of decreasing size, designated Bl
and B2. The partial separation of two RNA polymerase
activities by Strain et al. (47) would be consistent with
the presence of one less degenerate RNA polymerase, and
one in an advanced state of degeneration.
Enzyme Characterization
Maize RNA polymerase resembles the type II eukaryotic
RNA polymerases (1). The position of the maize RNA poly
merase in the DEAE-cellulose elution profile at 0.35 M
(NH^^SO^ (Figure 4) was as expected for a type II RNA
polymerase (32). Like the other type II enzyme (32), maize
RNA polymerase can utilize both native and denatured DNA
as a template (Table 4); it requires high salt (Figure 14)
2+ 2+
and either Mg of Mn as metal cofactors (Table 4). The
2+ 24-
strong preference shown for Mn over Mg by the sea urchin,

90
rat and calf thymus enzymes (32) was not observed for
maize RNA polymerase (Table 4). Rather than the 5-to
14-fold preference expected, the maize enzyme exhibited,
2+
at most, a 2-fold preference for Mn This low ratio
of Mn-dependent to Mg-dependent activity could be a unique
property of maize RNA polymerase, or it could reflect a
variable rate of polymerase degeneration. If the Mg-
dependent activity was more labile than the Mn-dependent
activity, then the low Mn/Mg activity ratio may have
resulted from the rapid purification procedure which per
mitted the maize Mg-dependent activity to remain active.
All the type II RNA polymerases have the unique property
of a-amanitin sensitivity (32). The evidence involving
enzyme purification and assay characteristics suggested,
and the inhibition by a-amanitin (Figure 21) confirmed
the inference, that the maize enzyme was a type II poly
merase (45). More importantly, a-amanitin inhibition
indicated poly(A) synthesis was also catalyzed by the
RNA polymerase II (Table 12). The synthesis of both RNA
and poly(A) by the same enzyme was supported by the co
purification of both activities during DAE-cellulose
chromotography, and the identical metal optima and the
identical K for ATP. Therefore, the same enzyme was
m
responsible for both activities.

91
RNA,and poly(A) synthesis by the same enzyme could
be distinguished by alterations in the reaction mixture
components since: 1. ATP was a substrate for both RNA
and homopolymer synthesis, while UTP was a substrate only
for RNA synthesis (Table 6). 2. Native DNA and denatured
DNA (Table 4), poly(dAT) and poly(dAdT) (Table 14), all
satisfied the template requirement for RNA synthesis,
but only denatured DNA and poly(dAdT) satisfied the tem
plate requirement for poly(A) synthesis. 3. The NTPs
were required for RNA synthesis (Figure 11), while poly(A)
synthesis was inhibited by low NTP concentrations (Fig
ure 19). 4. Actinomycin D inhibited RNA synthesis but
stimulated poly (A) synthesis (Table 12).
The synthesis of poly(A) with ATP, but not of
poly(U) with UTP, indicated the RNA polymerase synthesized
only one homopolymer. Synthesis of homopolymer required
nucleic acid (Table 4), and the nucleic acid was utilized
as a template and not as a primer (Table 14). Therefore,
poly(dT) regions must serve as templates to synthesize
poly(A) Since Watson-Crick base pairing would require
a poly(dT) template for poly(A) synthesis on DNA, as well
as on synthetic oligomers, DNA denaturation must increase
the exposed poly(dT) regions and provide the required
template for poly(A) synthesis.

92
Poly (A) and SNA are probably synthesized on the
same template. Addition of the NTPs increased AMP and
UMP incorporation equally (Table 6) reflecting RNA
synthesis. The increased AMP incorporation occurred above
the level of homopolymer synthesis measured when the NTPs
were absent. However, if very low. levels of NTPs were
added, poly (A) synthesis was inhibited (Figure 19). This
inhibition was identical to the NTP inhibition of E. coli
PJSTA polymerase during poly (A) synthesis by reiterative
transcription (46). The E. coli RNA polymerase was in
hibited when the enzyme began transcribing RNA with an
insufficient concentration of NTPs to support measurable
RNA synthesis. This resulted in a net inhibition of
AMP incorporation. Since delayed addition of low level
NTPs inhibited maize poly (A) and RNA synthesis, the syn
thesis of poly(A) arid SNA probably occurred on the same
DNA molecule. Utilization of the same DNA molecule for
poly(A) and RNA synthesis was further supported by the
actinomycin D resistance of poly(A) synthesis. In the
absence of the NTPs, poly(A) synthesis was refractory
to actinomycin D, but when NTPs were added, all incorpo
ration was inhibited by the actinomycin D (Table 12).
Since actinomycin D preferentially binds guanosine and
inhibits RNA synthesis by impeding the progress of the
enzyme along the DNA template (70), the poly(dT)-rich
regions of the DNA, unable to bind actinomycin D, would

93
remain available for reiterative poly(A) synthesis. With
NTP addition, reiterative transcription would cease, RNA
synthesis would begin, and the enzyme would encounter an
actinomycin D-guanosine complex and be inhibited. There
fore, both the NTP inhibition and actinomycin D inhibition
data are consistent with poly(A) and RNA synthesis occurring
on adjacent regions of the same DNA template.
Poly(A) and RNA are synthesized by the maize.RNA
polymerase II, and may be attached to one another. Maize
RNA polymerase synthesized RNA with native and with de
natured DNA, but the ApA frequency went from 30 percent
with native DNA (27) to 44 percent with denatured DNA
(Table 5). The increase in ApA frequency was not neces
sarily a change in the base composition of the RNA syn
thesized, but may have resulted from the simultaneous
synthesis of poly(A) and RNA on the denatured DNA template.
The early product synthesized in a complete reaction
mixture containing denatured DNA was resistant to
pancreatic ribonuclease, while late product was more
sensitive (Figure 23). This suggested the initial product
contained poly(A) and the subsequent product contained
RNA. Early poly(A) synthesis was also suggested by the
kinetics of AMP incorporation: a rapid initial rate in
which apparently all the nucleotides incorporated were
labeled; and a subsequent lower rate in which approximately
30 percent of the incorporated nucleotides were labeled

94
(Figured). Therefore, the rate of nucleotide elongation
appeared to be constant throughout poly(A) and RNA syn
thesis, although the rate of AMP incorporation changed.
If poly(A) synthesis preceths RNA synthesis, then poly(A)
would logically be expected at the 5' end of the RNA,
since the known RNA polymerases synthesize product from
the 5' to the 3' end (15). The linkage of poly(A) and
RNA, suggested by the data from maize, is strikingly
similar to that obtained with vaccinia virus cores by
Kates (53). Poly(A) synthesized by vaccinia cores
apparently was attached to RNA, but only when both were
synthesized simultaneously. With ATP alone, poly(A),
180 nucleotides long, was synthesized and net covalently
attached to RNA synthesized after the addition of the
NTPs. If both ATP and NTPs were initially present, poly(A),
50 nucleotides long, was synthesized and it was apparently
attached to the RNA (53). The data on maize poly(A) syn
thesis are consistent with the vaccinia system, therefore
long poly(A) sequences may be synthesized in the absence
of the NTPs, while short poly(A) sequences, attached to
RNA, may be synthesized in the presence of the NTPs.
Although resistance of products to RNase digestion resulting
from protein-product and DNA-product complexes (44) has
been ruled out in subsequent experiments, the attachment
of poly(A) to RNA has not been unequivocally demonstrated
in either the maize RNA polymerase reaction or the vaccinia
core reaction.

95
Poly(A) Synthesis and the Initiation
of Transcription
A major unsolved problem in eukaryotic transcription
is the initiation of RNA synthesis, including the recogni
tion of the binding site on DNA and selection of the
proper strand for transcription.. The discovery of poly (A)
synthesis by maize RNA polymerase II indicated the
phenomenon of reiterative poly(A) synthesis by prokaryotes
(E. cold.) could be extended to eukaryotes. Unlike the
prokaryotes, maize RNA polymerase II synthesizes poly(A)
and RNA during the same reaction. In prokaryotes, ATP
and GTP are found at the 5' terminus of newly synthesized
RNA. GTP initiation maybe an artifact of the in vitro
system (71), suggesting ATP is the first base incorporated
during initiation of transcription. Perhaps it is the
beginning of a short poly(A) strand, or perhaps the remnant
of a longer discontinuous poly(A) strand.
Transcription of native DNA is asymmetric (72), that
is, only one strand of the DNA at any one region of the
DNA is transcribed into RNA. If the RNA polymerase had
a binding affinity for only poly(dT)-rich regions on one
strand of the double helix, the complementary strand con
taining poly (dA) would not be bound. Therefore, not only
selection of the proper site, but selection of the proper
strand of DNA, might be attributed to poly(A) synthesis.

96
It ¡.is interesting to note that poly (A) is resistant
to rjbojatcslsase digestion. If poly (A) were involved in
the initiation of transcription, its metabolic stability
would insure transcription by forming a nuclease resistant
.initiation complex. Perhaps the reason that the denatured
DNA out-competed native DNA for the maize RNA polymerase
(Figure 7);r was that the maize polymerase had a greater
-affinity for the exposed poly(dT) regions. Since poly(dT)-
'rich regions would be most easily denatured (A:T pairs
are less strongly hydrogen bonded than G:C pairs), the
maize RNA polymerase would bind to the more accessible
regions of the partially denatured DNA. These RNA poly
merase binding sites could arise in vivo via the unwinding
proteins isolated by Alberts and Frey (73).
Both poly(A) and RNA are synthesized by the maize
RNA polymerase II and by E. coli RNA polymerase. Since
both enzymes can utilize a duplex DNA. template, and since
eukaryotic RNA polymerase may have a subunit structure
analogous to the paired structure of E. coli RNA polymerase
(x2 / 3 3 1) (see reference 11 and 38), there may be two
active sites (a,3) per RNA polymerase complex. One site
may recognize a binding site on one strand of the DNA tem
plate and synthesize poly(A) and RNA on that strand, while
the other site idles. Should the polymerase encounter an
initiation site on the opposite strand,, one site synthesizes
poly(A) and the other site the RNA, but sequentially and

97
in opposite directions. In APPENDIX B, a diagramatic
model.^foj the initiation of transcription is presented.
This model includes the initiation of RNA synthesis by
poly(A), the synthesis of RNA on either the same or
opposite DNA strands, and the presence of an RNA poly
merase containing two active sites, each site capable
of binding DNA and of polymerizing nucleotides.
The partially resolved peaks of two maize RNA
polymerase II activities reported by Strain et al. (47)
might reflect a degenerate RNA polymerase that can syn
thesize only RNA, and a less degenerate enzyme that can
synthesize both poly(A) and RNA. The apparent preference
of one RNA polymerase II for denatured DNA may represent
the synthesis of poly(A) by the less degenerate enzyme.
Similarily, the failure to detect homopolymer synthesis
by Stout and Mans (44), may reflect a similar degeneration
of the in vivo RNA polymerase complex. Maize RNA poly
merase purified by the procedure described in METHODS
does retain the ability to synthesize both poly(A). This
rapid purification procedure may be necessary to prevent
the degeneration of the RNA polymerase complex with the
associated decrease in activity, perhaps including the
loss of poly(A) synthesis. Further purification of the
maize RNA polymerase will determine whether such degenera
tion takes place, and whether the two polymerase types can

98
be detected; those that can synthesize only RNA and those
that can synthesize both poly(A) and RNA.

CONCLUSION
Maize DNA-dependent RNA polyirierase II synthesizes
both RNA and polyadenylic acid utilizing a DNA template.
The evidence is consistent with a model where polyadenylic
acid synthesis precedes the synthesis of RNA in the same
enzyme-template complex during the initiation of
transcription.
99

100
REFERENCES
- 1. .Cold Spring Harb. Symp. Quant. Biol. (1970). 35.
2. Silvestri, L. (ed.). Lepetit Colloquia on Biol. and
Med. (1969). American Elsevier Pub. Co., 52 Vander
bilt Ave., New York, N.Y.
3. Hanly, E. W. (ed.). RNA in Development, The Park City
Inter. Symp. on Prob. in Bio. (1969), Univ. of Utah
Press, Salt Lake City, Utah.
4. Burgess, R. R. (1971). Ann. Rev. Biochem., 40:711.
5. Von Hippel, P. H., and J. D. McChee (1972). Ann. Rev.
Biochem., 41:231
6. Losick, R. (1972). Ann. Rev. Biochem., 41:409.
7. Zubay, G. L. (ed.). Papers in' Biochemical Genetics,
1968). Holt, Rinehart and Winston, Inc., New York,
N. Y.
8. Watson, J. D. Molecular Biology of the. Gene., 2nd ed.
(1970). W. A. Benjamin, Inc., New York, N.Y.
9. Mahler, H. R. and Cordes, E. H. Biological Chemistry
(1966). Harper and Row, New York, N. Y.
10. Lehninger, A. L. Biochemistry (1971). Worth Publishers,
Inc., New York, N. Y.
11. Burgess, R. R., A. A. Travers, J. J. Dunn and E. K. F.
Bautz (1969). Nature, 221:43.
12. Losick, R. and A. L. Sonenshein (1969). Nature, 224:35.
13. Weiss, S. (1960). Proc. Nat. Acad. Sci., 46:1020.
14.Shorenstein, R. G. and R. Losick (1972). Fed. Proc.,
31:472. abs.

LO
i1
Chamberlin,
Biol., 35
M.
: 851.
(1970) .
Cold Spring Harb. Symp
16.
Roberts,
J.
W.
(1969) .
Nature, 224:1168.
17.
Travers,
A.
A.
(1970) .
Nature, 225:1009.

101
REFERENCES (continued)
.18.
Seifert, W., D. Rabussay and W.
'Lett., 16:175.
Sillig
(1970) FEBS
T9.
Goff, C. G. and K. Weber (1970). Cold
Symp. Quant. Biol., 35:101.
Spring Harb.
20.
Emmer, M., B. DeCrombrugghe, I.
(1970). Proc. Nat. Acad. Sci
Pastan and R. L. Perlman
., 66:480.
21.
Riggs, A. D. and S. Bourgeois
9.-A84.
(1969).
Biophys. J.,
22.
Jost, J. and H. V. Rickenberg
chem., 40:471.
(1971) .
Ann. Rev. Bio-
23.
Aronson, A. J. (1969). J. Mol
. Biol.,
11:576.
24.
Hussey, C., R. Losick and A. L.
J. Mol. Biol., 57:59.
Sonenshein (1971).
25.Zubay, G. D. Schwartz and J. Beckwith (1970). Proc.
Nat. Acad. Sci., 66:104.
26. Keller, W. and R. Goor (1970). Cold Spring Harb. Symp.
Quant. Biol., 35:671.
27. Stout, E. R. and R. J. Mans (1967). Biochem. Biophys.
Acta., 134:327.
28.Jacob, S. T., E. M. Sajdel and H. N. Munro (1968).
Biochem. Biophys. Acta., 157:421.
29. Seifart, K. H. and C. E. Sekeris (1967). Hoppe-
Seylers Z_. Physiol. Chem., 348 :1555 .
30. Cunningham, D. D., and D. F. Steiner (1967). Biochem.
Biophys. Acta., 145:834.
31. Roeder, R. G., and W. J. Rutter (1970). Proc. Nat.
Acad. Sci., 65:675.
32.Blatti, S. P., C. J. Ingles, T. J. Lindel, P. W. Morris,
R. F. Weaver, F. Weinberg, and W. J. Rutter (1970).
Cold Spring Harb. Symp. Quant. Biol., 35:649.
Hearst, J. E. and M. Botchan (1970). Ann. Rev. Biochem
39:151.
33.

102
REFERENCES (continued)
34.Teng, C. S., C. T. Teng and V. G. Allfrey (1971). J.
Biol. Chem., 246:3597.
35.Darnell, J. E. (1968). Bacteriol. Rev., 32:262.
36. Shearer, R., and B. J. McCarthy (1967). Biochem. J.,
6:283.
37. Blatti, S. P., personal communication.
38. Chambn, P., F. Gissinger, J. L. Mandel, C. Kedinger,
M. Gniazdowski and M. Maihlac (1970). Cold Spring
Harb Symp. Quant. Biol., 35:693.
39. Kedinger, C. P. Nuret and P. Chambn (1971). FEBS Lett.,
1_5: i69 .
40. Huang, R. C., N. Maheshwari and J. Bonner (1960).
Biochem. Biophys. Res. Comm., 3:6.
41. Bonner, J., and R. C. Huang (1966). Biochem. Biophys.
Res. Comm.. 22:211.
42. Horgen, P. A., and J. L. Key (1972). Plant Physiology,
49:13.
43. Mans, R. J., and G. D. Novelli (1964). Biochem. Bio-
phys. Acta., 91:186.
44. Stout, E. M., and R. J. Mans, Plant Physiology, 43:405.
45. Benson, R. H. (1971). Plant Physiology, 47:36abs.
46. Chamberlin, M. and P. Berg (1964). J. Mol. Biol., 8:
708.
47. Strain, G. C., K. P. Mullinix and L. Bogorad (1971).
Proc. Nat. Acad. Sci., 68:2647.
48. Edmonds, M. and R. Abrams (1962). J. Biol. Chem.,
237:2636.
49. Klemperer, H. G. (1965). Biochem. Biophys. Acta., 95:
251.
50. Mans, R. J., and T. J. Walter (1971). Biochem. Biophys.
Acta., 247:113.

103
REFERENCES (continued)
51. Mans, R. J. (1971). Biochem Biophys. Res. Comm.,
45:980. "
52. Hurwitz, J., J. J. Furth, M. Anders and A. Evans
(1962). J. Biol. Chem., 237:3752.
53.Kates, J. (1970). Cold Spring Harb. Symp. Quant.
Biol., 35:743. -
54. Edmonds, M., M. H. Vaughan, Jr., and H. Kakazato (1971)
Proc. Nat. Acad. Sci. 68:1336.
55.
Darnell, J. E., R.
Proc. Nat. Acad.
Wall
Sci. ,
and R. J. Tushinski (1971).
£8:1321.
56.
Lee, S. Y., J. Mendecki,
Proc. Nat. Acad. Sci.,
and G. Brawerman
68:1331.
(1971).
57.
Green, M., and M.
£9:791.
Cartas
(1972). Proc.
Nat. Acad. Sci.
58.
Penman, S. H, M..
Roshas
h and S. Perlman
(1970), Proc.
Nat. Acad. Sci., 67:1878.
59. Lim, Y., and E. S. Cannelakis (1970). Nature, 227:710.
60. Burr, H. and J.. B. Lingrel (1971). Nature N. B.,
233:41.
61. Heaust, J. E., and M. Botchan (1970). Ann. Rev. Bio
chem. 39:177.
62. Lowry, O. H., N. J. Rosenbough, A. L. Farr and R. J.
Randall (1951). J. Biol. Chem., 193:265.
63. Hulbert, R. B., in S. P. Colowick and N. 0. Kaplan
(ed.)f Methods in Enzymology, 3_:21a.
64. Davis, B. J. (1964). New York Academy Science, 121:404
65. Weber, K., and M. Osborn (1969). J. Biol. Chem., 244:
4406.
66. Peterson, E. A., and H. A. Sober, in S. P. Colowick and
N. 0. Kaplan (ed.) Methods in Enzymology, £:3.
67. Burgess, R. R. (1969). J. Biol. Chem., 244:6168.
68.Gellespie, D., and. S. Speigelman (1965). J. Mol. Biol.
12:820.

104
REFERENCES (continued)
69. Benson, R. H., and R. J. Mans (1972).
427abs.
70. Richardson, J. P. (1966). J. Mol. Biol.
71. Jacobson, A., and D. Gillespie (1970).
Harb. Symp. Quant. Biol., 35:85.
72. Summers, W. C., and W. Szybalski (1968)
34:9.
Fed. Proa, 31
, 21:83.
Cold Spring
Virology,
73.Albert, B. M., and L. Frey (1970). Nature, 227:1313.

APPENDIX A
AMP Incorporation and ATP Specific Activity
All activity measurements were based on the
incorporation of radioactivity into an acid-insoluble
product. Since the products formed by the RNA polymerase
contained AMP [8~-^C]ATP was used as the labeled precursor.
The fraction of the product AMP labeled should, therefore,
be proportional to the ATP specific activity. To test this
assumption, the specific activity of the ATP was varied
from 2.2 to 6.5 yCi/ymole, at three different ATP concen
trations: 0.25 mM, 0.5 mM and 1.0 mM. The amount of
acid-insoluble radioactivity synthesized in a standard
reaction mixture was found to be directly proportional to
the ATP specific activity (Figure 24). The predicted re
lationship between AMP incorporation and the specific
activity of the ATP substrate was, therefore, confirmed.
Comparisons of enzymatic activities could now be made,
even if the ATP specific activity was varied, provided
enzyme activity was expressed as pmoles of AMP incorporated.
When a more radioactive product is needed, such as
during product characterization and hybridization procedures,
the cpm incorporated into the product can be increased
with additional labeled ATP. This can be achieved by
keeping the amount of labeled ATP constant and decreasing
105

106
ATP SPECIFIC ACTIVITY C/M
Figure 24. AMP INCORPORATION AS A FUNCTION OF
ATP SPECIFIC ACTIVITY
Assayed in a standard reaction mixture containing
1.0 ymole MgCl-, 0.37 A 26q native DNA and 4 yg RNA
polymerase. Tne ATP concentrations were as follows:
1.0 mM (solid circles), 0.5 mM (solid squares) and
0.25 mM (solid triangles).

107
tlie amount of cold ATP added. The net effect is an
increase in the specific activity of the labeled ATP.
However, since the ATP concentration is no longer satu
rating the reaction, the total pmoles of AMP incorporated
decreases. This decrease in incorporation with lower ATP
concentrations was a consistent percentage for all time
points examined out to 40 min. (Figure 25). If the ATP
concentration was decreased from 1.0 mM to 0.5 mM, 86 per
cent of the maximum rate remained; if the ATP concentration
was decreased to 0.25 mM, 68 percent of the maximum rate
remained. Since the labeled ATP was held constant as the
total ATP concentration was decreased there was a 2-fold
increase in specific activity with 0.5 mM ATP, and a 4-fold
increase with 0.25 mM. The. combined result of the decreased
amount of AMP incorporated and the increased labeling of
that which was incorporated was a 72 percent increase in
cpm for 0.5 mM ATP and a 164 percent increase in cpm for
0.25 mM ATP. Therefore, the labeled substrate was conserved
and more cpm were incorporated into the RNA polymerase
product.

to
Assayed in a standard reaction mixture containing 1.0 ymole MgCl, 0.37 Anative DNA and
4 yg RNA polymerase. The 14C-ATP specific activity was as follows: panel A; 6.5 Ci/mole,
panel B; 4.5 Ci/mole, panel C; 2.1 Ci/mole. The ATP concentrations were as follows: 1.0 mM
(solid circles), 0.5 mM (open circles) and 0.25 mM (solid triangles).
108

APPENDIX B
A Model for Polyadenylic Acid Initiation
of Transcription
Enzyme Structure
The RNA polymerase consists of a core enzyme with
paired subunits (a2, 82)* Additional factors or subunits
may confer template specificity or additional activities
upon this core polymerase. Each pair of subunits (a,8)
of the core enzyme contains the ability to bind DNA and
to catalyze ENA synthesis, therefore, creating the
potential for two active sites per RNA polymerase complex
(Figure 26) .
Enzyme Activity
Properties of Site 1
Site 1 synthesizes RNA and will bind Randomly on
2+ 2+
denatured DNA. It will utilize either Mg or Mn and
it will incorporate all four NTPs. It is sensitive to
cs-amanitin and actinomycin D. It will actively catalyze
RNA for more than 90 min. in vitro.
109

110
I
n
Figure 26. A MODEL FOR POLYADENYLIC ACID
INITIATION OF TRANSCRIPTION
I- Polyadenylic acid and RNA transcribed
from the same strand of DNA.
Polyadenylic acid and RNA transcribed
from opposite strands of DNA.
II.

Ill
Properties of Site 2
'synthesizes poly (A) and RNA, depending upon
the assay substrate and template. It binds selectively
to poly(dT) regions of denatured DNA and when synthesizing
2+
poly(A) has a strong preference for Mn as a cofactor.
In the presence of ATP it will reiteratively tran
scribe the short poly(dT) template and synthesize long
poly(A) sequences. In the presence of the NTPs, it will
synthesize a short poly(A) sequence and then begin
transcribing RNA. At low NTP concentrations, site 2
begins transcribing into the DNA and is inhibited. It
is inhibited by a-amanitin, but resistant to actinomycin
D wnen sy'Ci'iasj.i.mg pOj_y \1\) .
This model for initiation of transcription on
double stranded DNA is based upon data with denatured
DNA. The presence of a mechanism for selectively de
naturing the initiation region must, therefore, be
present iri vivo.
It is suggested the poly(dT)-rich template region
of the denatured DNA may be in a circular helix, permit
ting reiterative transcription and poly(A) synthesis
to occur.

BIOGRAPHICAL SKETCH
Robert Henry Benson legally began to exist on
December 15, 1942 in Chicago, Ill. He lived with his
parents, Henry and Mary Anne Benson, and four brothers,
Gary, John, Richard and Kennith, until graduation from
Miami Jackson High School in 1961. He was forced to
leave home to attend the University of Florida (1961-62,
1964-67) and the U. S. Coast Guard Academy (1962-64),
graduating from the University of Florida in 1967 with a
bachelor's degree in chemistry. From 1967 to 1972, he
was cared for by the N. I.H. through the Department of
Immunology and Medical Microbiology. In the Summer of
1969 he met Rusty J. Mans who has kept him busy ever since.
During a period of sensual insanity in October 1970, he
and Barbara A. Mendheim were married and they have one
Dachshund, Bernardine, age 2.
112

I certify that I have read this study and that in
ray opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Rusty Jjl^ans, Chairman
Prq^'essor of Biochemistry
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Daniel Billen
Professor of Radiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
John W. Cramer
dissociate Professor of
Pharmacology and Therapeutics
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree, of Doctor of Philosophy.
George E. Gifford
Professor of Immunology and
Medical Microbiology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation forthe degree of Doctor of Philosophy.
JL
Ira G. Rosen
Assistant Professor
Immunology and Medical
Microbiology
This dissertation was submitted to the Dean of the
College of Medicine and to the Graduate Council, and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.
Dean, Graduate School

2) <4-14 p
health
center
library.



TABLE 11
AVERAGE CHAIN LENGTH OF POLYADENYLIC ACID
A standard reaction mixture was used containing 0.037 A26Q
denatured DNA, 0.5 ymoles MnCl2/ no NTPs and 12 yg RNA
polymerase. The reaction was incubated for 60 min. at 30.
Yeast RNA (1 mg) was added and the product precipitated by
the addition of 0.3 ml of cold 1.0 M HC10^ containing 5 mM
sodium pyrophosphate. The ppt. was washed 4X with 1 ml of
cold 0.5 M HC10^ and then alkaline hydrolyzed and
electrophoresised (see METHODS).
Compound CPM
AMP
2,200
Adenosine
<22


10
First, transcription may be activated by removal of histones,
somewhat analogous to removal of the repressor on the lactose
operon. Second, RNA polymerase and specific initiation
factors initiate transcription by opening the genes for
transcription, with histone removal occurring as the RNA
polymerase precedes (15). Accordingly, either the histones
or the RNA polymerases may function to control the specificity
of transcription, or a combination of both systems.
Acidic nuclear proteins are of interest to transcription
because of their physical properties and location (34).
Since the RNA polymerases are themselves acidic nuclear
protein complexes, some of the acidic proteins may be sub
units of the RNA polymerases or of the other polymerases.
The acidic nuclear proteins may contain factors that function
along with the histones for gene selection and control of
transcription.
Transcription Products
There are at least three classes of RNA produced in
all cells: rapidly labeled RNA including HnRNA and mRNA;
stable RNA or GC-rich RNA which is rRNA; and soluble or
tRNA. These three classes of RNAs constitute the bulk of
the transcriptional products in all cells. Following
transcription, the RNA is often processed by specific
systems which selectively degrade the gene product into
the functionally active form. The processing of ribosomal
T


9
purifisaatlo-x^WAS. vex^. slow. This was a. result of the
initial |.o^'avti^ty :of the RNA polymerase in eukaryotic
tissues, of fcfej&f&iiwf-i-culty encountered in freeing the enzyme
of contaminating 'DNA and of the instability of the enzyme
during purification (26) These problems were overcome and
soluble DNA-dependent RNA polymerases were purified from
eukaryotic tissues (27,28,29,30). In 1970, Roeder and
Rutter (31) first detected the presence of two types of
RNA polymerase--5:' Hype I was a nucleolar RNA polymerase,
insensitive to a-amanitin, that synthesized a product which
competitively-..hybridized with r-RNA but not with Hn-RNA.
Type II was a nupl^oplasmic, a-amanitin sensitive RNA poly
merase that synthesized a product that competitively
hybridized with Hn-RNA but not with r-RNA (32). This was
the first time that specific RNA polymerases were shown
to be localized inside a eukaryotic cell and to be re
sponsible for specific classes of eukaryotic RNA. Since
then it has been shown that the nucleus, the nucleolus,
and the cellular organelles have unique transcriptional
systems (15).
Eukaryotes have a complex chromosomal structure
involving nucleic acids, histones, and acidic proteins.
The relationship between the histones and differentiation
is. uncertain, although histones are believed to be inti
mately involved 'in gene selection (33). There are two
major approaches to how this interaction might occur.


106
ATP SPECIFIC ACTIVITY C/M
Figure 24. AMP INCORPORATION AS A FUNCTION OF
ATP SPECIFIC ACTIVITY
Assayed in a standard reaction mixture containing
1.0 ymole MgCl-, 0.37 A 26q native DNA and 4 yg RNA
polymerase. Tne ATP concentrations were as follows:
1.0 mM (solid circles), 0.5 mM (solid squares) and
0.25 mM (solid triangles).


74
whereas the radioactivity detected in the adenosine fraction
did not exceed background (Table 11). The sensitivity of
the assay for adenosine was 22 cpm; therefore the lower
estimate of the average chain length for poly(A) was 2,200/
22, or greater than 100 nucleotides per chain after 60 min.
at 30.
Summary
Maize RNA polymerase synthesized polyadenylic acid in
the presence of denatured DNA, ATP and metal, preferably
2+
Mn There was greater than a 17-fold preference for
ATP over UTP as substrate for homopolymer synthesis. Low
levels of the NTPs as a mixture or added individually in
hibited poly(A) synthesis, independent of the time of
addition. Poly(A) synthesis was directly proportional to
the amount of enzyme protein added. The product remained
acid-insoluble after boiling with.. SDS. The average chain
length of the product accumulated after 60 min. incubation
was greater than 100 nucleotides and the ApA frequency was
0.90.
Comparison of RNA and Polyadenylic
Acid Synthesis
The two AMP incorporating activities measuring RNA
and poly(A) synthesis were apparently catalyzed by the same
enzyme; RNA polymerase II (69). The evidence for this


52
with ATP above 1.0 mM (Figure 12). A Lineweaver-Burke
. -4
reciprocal plot resulted in a of 1.25 x 10 M ATP
(Figure 13). The Km was the same with native and dena
tured DNA, although the V with denatured DNA was twice
that with native DNA. The standard ATP concentration for
the RNA polymerase reaction mixture was 1.0 mM, since it
resulted in the highest ATP specific activity while main
taining the maximum rate of RNA synthesis. The rate of
AMP incorporation decreased when the ATP concentration was
below saturation. If the ATP concentration was decreased
to 0.5 mM there was 86 percent of the maximum rate and if
it was decreased to 0.25 mM there was 68 percent of the
maximum rate. The pmoles of AMP incorporated per reaction
was independent of the [8-l4C]ATP specific activity (see
APPENDIX A.).
Salt
As with the partially purified enzyme, the more
highly resolved preparations exhibited a dependency on
added (NH^^SO^ for RNA polymerase activity, especially
in the presence of native DNA (Figure 14). The maximum
level of activity occurred between 60 and 90 mM (NH4)2S04
with native DNA, and up to 100 mM with denatured DNA.
Because the RNA polymerase fraction contained 0.4 M
(NH^)2S0^, the titrations at concentrations below 40 mM
were not performed.


BIOGRAPHICAL SKETCH
Robert Henry Benson legally began to exist on
December 15, 1942 in Chicago, Ill. He lived with his
parents, Henry and Mary Anne Benson, and four brothers,
Gary, John, Richard and Kennith, until graduation from
Miami Jackson High School in 1961. He was forced to
leave home to attend the University of Florida (1961-62,
1964-67) and the U. S. Coast Guard Academy (1962-64),
graduating from the University of Florida in 1967 with a
bachelor's degree in chemistry. From 1967 to 1972, he
was cared for by the N. I.H. through the Department of
Immunology and Medical Microbiology. In the Summer of
1969 he met Rusty J. Mans who has kept him busy ever since.
During a period of sensual insanity in October 1970, he
and Barbara A. Mendheim were married and they have one
Dachshund, Bernardine, age 2.
112


20
there were 5 adenosine residues for every adenosine
tetraphosphate (53). Therefore, if poly(A) was the initial
sequence and contained the tetraphosphate, there were four
more sequences that were synthesized internally or at the 3'
terminis.
Based upon hybridization of poly(A) to denatured
vaccinia DNA, Kates estimated there could be greater than
25 poly(dT) sequences in the vaccinia DNA, each 180
nucleotides long (53). This wa consistent with Heaust
and Botchan (61), who stated that 10 percent of the genome
of mice consisted of AT-rich regions. The function of this
DNA was not clear, but it was probably not transcribed in
vivo (61). It was, however, known to be uniformly distri
buted among all chromosomes.
Summary
Poly(A) sequences are known to occur in vivo and in
vitro. They appear to be synthesized by two enzyme systems,
poly(A) polymerases sensitive to cordycepin, and by RNA
polymerases insensitive to cordycepin. Some poly(A) se
quences are located at the 3" hydroxyl end of RNA polymers;
however, there is evidence for both poly(A) sequences in
ternally and at the 5' end of the RNA. The importance and
functional significance of these sequences is not firmly
established.


APPENDIX A
AMP Incorporation and ATP Specific Activity
All activity measurements were based on the
incorporation of radioactivity into an acid-insoluble
product. Since the products formed by the RNA polymerase
contained AMP [8~-^C]ATP was used as the labeled precursor.
The fraction of the product AMP labeled should, therefore,
be proportional to the ATP specific activity. To test this
assumption, the specific activity of the ATP was varied
from 2.2 to 6.5 yCi/ymole, at three different ATP concen
trations: 0.25 mM, 0.5 mM and 1.0 mM. The amount of
acid-insoluble radioactivity synthesized in a standard
reaction mixture was found to be directly proportional to
the ATP specific activity (Figure 24). The predicted re
lationship between AMP incorporation and the specific
activity of the ATP substrate was, therefore, confirmed.
Comparisons of enzymatic activities could now be made,
even if the ATP specific activity was varied, provided
enzyme activity was expressed as pmoles of AMP incorporated.
When a more radioactive product is needed, such as
during product characterization and hybridization procedures,
the cpm incorporated into the product can be increased
with additional labeled ATP. This can be achieved by
keeping the amount of labeled ATP constant and decreasing
105


AMP INCORPORATED pmoles
51
Figure 11. NTP TITRATION OF RNA POLYMERASE
Assayed in a standard reaction mixture con
taining 1.0 uniles MgC^/ 0.37 nat:*-ve
DNA, 6.4 yg RNA polymerase and pooled NTPs
(GTP, CTP, UTP) as indicated.


72
TABLE 10
NEAREST NEIGHBOR FREQUENCY ANALYSIS OF
POLYADENYLIC ACID PRODUCT
A standard reaction mixture containing 0.37 A2gQ denatured
DNA, 0.10 ymoles [a-32p]ATP (specific activity 30 mCi/mmole) ,
0.4 ymoles MnCl2 and 10 yg enzyme was incubated at 30 for
60 min. 1% SDS was added to the reaction, it was passed
over sephadex G25, material in the excluded volume was
hydrolyzed in 0.3 M KOH for 18 h at 37, adsorbed to acti
vated charcoal and the eluted nucleotides lyophilized, re
suspended in H20 and resolved by paper electrophoresis (see
METHODS),
CPM
Added
Recovered
a
CpA ApA GpA UpA
11,000 4,500 0.01 0.90 0.04 0.05
iSome of the sample was lost during lyophilization.


11
RNA is the best studied. The ribosomal genes are
sequestered.- in the nucleolus and are transcribed as a unit
into a singde ?40-45 S precursor RNA molecule. This RNA is
pxocessed by a series of post-transcriptional cleavage steps
to give 18 S and 25 S RNAs found in mature amphibian oocyte
ribosomes (35). Heterogeneous RNA is found in the nucleo
plasm and has a DNA-like base composition. A large fraction
of this rapidly labeled RNA never leaves the nucleus and
may be involved with regulation at the level of transcription
(36). Both rRNA and tRNA are stable and constitute the
bulk of the RNA contained in a cell at any one time, while
mcvst of the- rRNA rapidly turns over.
RNA Polymerases
Types
Type I RNA polymerase is localized in the nucleolus
and synthesizes GC-rich RNA that competitively hybridizes
with ribosomal RNA [32). Its polymerizing activity is
resistant to ct-amanitin, it is highly sensitive to actinomycin
D and it is refractory to rifampicin (32). On DEAE-sephadex
it is the first RNA polymerase eluted by a linear ammonium
sulfate gradient, generally around 0.2 M (NH^^SO^. The
type I RNA polymerase has a preference for magnesium and
low salt concentrations (0.04-0.07 M (NH^^SO^). The type I
RNA polymerase is very unstable and therefore difficult to
purify (37).


LIST OF TABLES
1. STEPS IN RNA POLYMERASE PURIFICATION 32
2. STORAGE AND FREEZE-THAW LABILITY OF RNA
POLYMERASE 40
3. SALT PRECIPITATION OF RNA POLYMERASE 42
4. ASSAY REQUIREMENTS OF RNA POLYMERASE 44
5. NEAREST NEIGHBOR FREQUENCY ANALYSIS OF RNA
PRODUCT SYNTHESIZED WITH DENATURED DNA. 59
6. AMP AND UMP INCORPORATION BY RNA POLYMERASE . .65
7. INHIBITION OF POLYADENYLIC ACID SYNTHESIS BY
NUCLEOTIDES . 67
8. EFFECT OF DELAYED ADDITION OF NTPs ON POLYADENYLIC
ACID SYNTHESIS. .68
9. EFFECT OF HEATING' AND SDS ON RNA POLYMERASE
PRODUCTS 71
10. NEAREST NEIGHBOR FREQUENCY ANALYSIS OF POLY
ADENYLIC ACID PRODUCT .72
11. AVERAGE CHAIN LENGTH OF POLYADENYLIC ACID 73
12. INHIBITOR SENSITIVITY OF RNA POLYMERASE 76
13. COMPARISON OF CALF THYMUS AND MAIZE DNA AS
TEMPLATES FOR RNA POLYMERASE 82
14. RNA POLYMERASE ACTIVITY WITH SYNTHETIC TEMPLATES. .84
vi


AMP INCORPORATED pmelsG
55
t
... t I
ICO
AMMONIUM SULFATE mM
Figure 14. AMMONIUM SULFATE TITRATION OF
RNA POLYMERASE
Assayed in standard reaction mixture con
taining 1.0 ymole MgCl^> -37 &260 DNA'
either native (solid circles) or denatured
(solid triangles), 6.4 yg RNA polymerase and
(NH^^SO^ as indicated.
200


ABSORBANCE AT 280nm
Figure 3. DEAE-CELLULOSE ELUTION PROFILE
AMMONIUM SULFAT


19
Tiie strongest evidence for poly (A) being contained
in newly synthesized RNA was that of Kates (53) Utilizing
' vacciiiiV cores incubated in vitro, under conditions of RNA
synthesis, Kates'data indicated that after mild RNase treat
ment all of the RNase resistant poly(A) sequences sediment-
ated at 4 S and had a chain length greater than 50 nucleo
tides. The association of poly(A) with RNA was not dis
rupted by heating at 100, nor by 75 percent DMSO at 80
,in the presence of cold poly(A). This indicated that, if
in fact, the poly (A) was attached to the RNA it was probably
through a covalent bond. When poly(A) was synthesized by
vaccinia cores with only ATP as the substrate, then the
poly(A) was not later attached to newly synthesized viral
RNA (53). This in vitro poly(A) synthesis continued for
only 5 min., the poly(A) was 180 nucleotides long and had
a uniform 5.8 S value. After alkaline hydrolysis and
chromotography there was one 5' tetraphosphate and one
adenosine for every 180 AMP residues (53). If synthesis
'-occurred in the presence of all 4 nucleoside triphosphates,
25-30 percent of the AMP incorporated was in poly(A). The
poly(A) was synthesized without a lag period upon addition
of the substrates, but if UMP incorporation was measured,
there was a 1.5 min. lag before incorporation began (53).
This indicated poly(A) synthesis preceded RNA synthesis.
If the poly(A) synthesized in the presence of all 4 nucleo
side triphosphates was purified and alkaline hydrolyzed,


56
Enzyme
If enzyme protein was not added there was no AMP
incorporation in the standard RNA polymerase reaction
mixture (Table 4). The total AMP incorporated in 20 min. was
the function of the enzyme protein added, up to 6 yg pro
tein per standard reaction mixture .(Figure 15). In the
presence of the NTPs, the amount of AMP incorporated with
denatured DNA.was twice that incorporated with native DNA,
at all enzyme concentrations.
Rate of AMP Incorporation
The amount of AMP incorporated in a standard RNA
polymerase reaction increased as a function of time for
more than 90 min. (Figure 16). The rate of AMP incorpora
tion decreased continuously during the initial 20 min. of
incubation and then became constant. The initial rate
(R^) and the late rate (R£) were arbitrarily defined as
that occurring up to 20 min., and that occurring after
20 min. What appeared to be two different reactions
(initial and late) were not resolved by increasing the
incubation temperature from 30 to 37.
Nearest Neighbor Frequency
Product synthesized on denatured calf DNA with
[a32p]ATP after alkaline hydrolysis, yielded four labeled
nucleotides (Table 5) Since the product was acid-insoluble
and excluded from sephadex G25, indicating an oligomer,
the nearest neighbor frequency data indicated the product


12
The type II RNA polymerase is a nucleoplasmic RNA
polymerase that synthesizes a product which competitively
hybridizes with HnRNA. (32). Its activity is sensitive
to a-amanitin and it is the second RNA polymerase eluted
by a linear (NH^^SO^ gradient from DEAE-sephadex, generally
around 0.3 M salt. The type II enzyme exhibits more
activity with manganese, rather than with magnesium, and
is most active at high salt concentrations (0.9-0.12 M
(NH^^SO^) (32). It is more stable than the type I RNA
polymerase, but is inactivated easily, particularly during
salt precipitation.
Type.Ill RNA polymerase is a nucleoplasmic RNA
polymerase, eluted third on DEAE-sephadex chromotograph,
generally around 0.35 M (NH4)2SC>4 (32). The small amount
of type III enzyme activity present in extracts is often
undetected. The type III RNA polymerase is resistant to
a-amanitin, prefers manganese and has a broad salt optimum
(0-0.2 M (NH4)2SC>4) (32). It is the least studied of the
nuclear RNA polymerases and it has been proposed that it may
synthesize tRNA (32) .
The type IV RNA polymerase, refers to RNA polymerase
activity detected in cellular organelles, specifically
chloroplasts and mitochondria. Little is known of these
enzymes due to the difficulty in extracting the quantity of
material necessary for enzyme purification, and to the
difficulty in removing the other contaminating RNA polymerases.
4


TABLE OF CONTENTS
Acknowledgments iii
List of Tables vi
List of Figures vii
Key to Abbreviations ix
Abstract xi
Introduction 1
Literature Review 4
Methods and Materials. . 21
Results 31
Discussion 36
Conclusion 99
References 100
Appendix A 105
Appendix B 109
Biographical Sketch 112


45
DNA
,iv 2+
^fte';'.c'orpe'te system with Mg as the metal cofactor,
the-'B'NA' requirement- was satisfied with either native or
heat-denatured DNA (Table 4). The reaction was saturated
with denatured DNA in excess of 0.4 A260//m'*"' whereas with
native DNA more than a 5-fold higher concentration (1.8 A2g(/
ml) was required to reach the same level of activity (Fig
ure 7). If equal amounts of native and denatured DNA were
combined and /assayed in the same reaction mixture, the
activity measured could be accounted for by the denatured
DNA alone* Thus, the denatured DNA appeared to out-compete
the native DNA ;,:for the RNA polymerase. However, at
saturating concentrations native and denatured DNA supported
equivalent AMP incorporation.
If MnCl2 was substituted for MgCl2 in the reaction
mixture, there was almost a 2-fold increase in RNA polymerase
activity with denatured DNA (Table 4). In the presence of
2+
Mn the activity with denatured DNA was twice that with
native DNA over a range of DNA concentrations (Figure 8).
Although the RNA polymerase assay was saturated at 0.4 A^g/
ml with both native and denatured DNA, AMP incorporation
after 20 min. was not equivalent. Therefore, the concen
tration dependence of the polymerase on native and denatured
2+
DNA differed when assayed with Mg but the maximum level
2+
of activity'was the same. In contrast with Mn the con
centration dependence with native and denatured DNA was


ABSORBANCE AT 280nm
Figure 4. RNA POLYMERASE ACTIVITY IN
DEAE-CELLULOSE FRACTIONS
Assayed in a standard reaction mixture
containing 0.5 nmoles MnCl9, 0.037 A260
DNA; either denatured (solid triangles)
or native (open triangles) and *2t to 4 yg
enzyme.
AMP INCORPORATED nmoles


94
(Figured). Therefore, the rate of nucleotide elongation
appeared to be constant throughout poly(A) and RNA syn
thesis, although the rate of AMP incorporation changed.
If poly(A) synthesis preceths RNA synthesis, then poly(A)
would logically be expected at the 5' end of the RNA,
since the known RNA polymerases synthesize product from
the 5' to the 3' end (15). The linkage of poly(A) and
RNA, suggested by the data from maize, is strikingly
similar to that obtained with vaccinia virus cores by
Kates (53). Poly(A) synthesized by vaccinia cores
apparently was attached to RNA, but only when both were
synthesized simultaneously. With ATP alone, poly(A),
180 nucleotides long, was synthesized and net covalently
attached to RNA synthesized after the addition of the
NTPs. If both ATP and NTPs were initially present, poly(A),
50 nucleotides long, was synthesized and it was apparently
attached to the RNA (53). The data on maize poly(A) syn
thesis are consistent with the vaccinia system, therefore
long poly(A) sequences may be synthesized in the absence
of the NTPs, while short poly(A) sequences, attached to
RNA, may be synthesized in the presence of the NTPs.
Although resistance of products to RNase digestion resulting
from protein-product and DNA-product complexes (44) has
been ruled out in subsequent experiments, the attachment
of poly(A) to RNA has not been unequivocally demonstrated
in either the maize RNA polymerase reaction or the vaccinia
core reaction.


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation forthe degree of Doctor of Philosophy.
JL
Ira G. Rosen
Assistant Professor
Immunology and Medical
Microbiology
This dissertation was submitted to the Dean of the
College of Medicine and to the Graduate Council, and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.
Dean, Graduate School


26
(NH^^SO^; 0.37 calf thymus DNA; 5 yg BSA and enzyme
as indicated (27). An assay mixture was prepared immediately
before use and held on ice until the reaction was initiated
by the addition of enzyme to the mixture. The reaction
tube was immersed in a 30 water bath for 20 min. and acid-
insoluble radioactivity was determined by a modification of
a procedure previously described (27). The reaction was
terminated by pipetting the mixture onto a 3 MM filter
paper disk. The reaction mixture was absorbed into the
disk for 10 sec. under a heat lamp and precipitated by
immersion in cold 10 percent TCA containing 2 mM sodium
pyrophosphate. As many as 40 disks were then extracted
five times with 100 ml of the TCA solution (5 min. each
time), once with 100 ml ethanol-ether (1:1), and once with
100 ml ether for 3 to 5 min. All extractions were per
formed at room temperature. The disks were then dried
and counted in a scintillation solution containing PPO,
POPOP and reagent grade toluene. Disks which received
reaction mixture containing no enzyme averaged 15 cpm
above machine background. The wash procedure reduced the
cpm from approximately 400,000 cpm/disk to 35 cpm/disk
for the no enzyme control. Machine counting efficiency
was 0.75 cpm/dpm. One unit of RNA polymerase activity is
defined as 1 nmole AMP incorporated into acid-insoluble
material under standard reaction mixture conditions in
20 min.


40
TABLE 2
STORAGE AND FREEZE-THAW LABILITY OF RNA POLYMERASE
After the indicated treatment aliquots of one enzyme
preparation were assayed for RNA polymerase activity in a
standard reaction mixture containing 1.0 pmole MgClj,
0.37 A.26Q native DNA and 18 yg enzyme. 100% incorporation
was equivalent to 172 pmoles AMP incorporated per reaction.
% Incorporated
A) Temperature (19 days)
-76 (Reveo freezer) 98
-17 (Freezer) 1
B) Glycerol Storage5 (17 h)
-196 (Liquid nitrogen) 96
-17 (Freezer) 78
4 (Refrigerator) 48
b
C) Freeze-thaw^
One 100
Two 100
Three 86
Four 82
Five 79
Storage on Ice (5 h)C 80
aGlycerol was added to 20% by volume to the enzyme preparation
just prior to storage.
A. vial of enzyme frozen in liquid nitrogen was thawed at 37
and held on ice prior to assay. After assay it was refrozen
in liquid nitrogen. This sequence was repeated as indicated.
CEnzyme frozen and thawed once prior to 5 h storage on ice.


96
It ¡.is interesting to note that poly (A) is resistant
to rjbojatcslsase digestion. If poly (A) were involved in
the initiation of transcription, its metabolic stability
would insure transcription by forming a nuclease resistant
.initiation complex. Perhaps the reason that the denatured
DNA out-competed native DNA for the maize RNA polymerase
(Figure 7);r was that the maize polymerase had a greater
-affinity for the exposed poly(dT) regions. Since poly(dT)-
'rich regions would be most easily denatured (A:T pairs
are less strongly hydrogen bonded than G:C pairs), the
maize RNA polymerase would bind to the more accessible
regions of the partially denatured DNA. These RNA poly
merase binding sites could arise in vivo via the unwinding
proteins isolated by Alberts and Frey (73).
Both poly(A) and RNA are synthesized by the maize
RNA polymerase II and by E. coli RNA polymerase. Since
both enzymes can utilize a duplex DNA. template, and since
eukaryotic RNA polymerase may have a subunit structure
analogous to the paired structure of E. coli RNA polymerase
(x2 / 3 3 1) (see reference 11 and 38), there may be two
active sites (a,3) per RNA polymerase complex. One site
may recognize a binding site on one strand of the DNA tem
plate and synthesize poly(A) and RNA on that strand, while
the other site idles. Should the polymerase encounter an
initiation site on the opposite strand,, one site synthesizes
poly(A) and the other site the RNA, but sequentially and


33
AMP/mg}', however, it was more dilute (0.2 mg/ml) and its
preparation more time consuming (6 h). Batch eluted RNA
polymerase had a lower peak specific activity (90 nmoles AMP/
mg), but it was more concentrated (0.5 mg/ml), more rapidly
eluted (2 h) and the net recovery was better than with the
gradient procedure.
The profile of &2QQ materaal during gradient elution
of the DEAE-cellulose column indicated most of the material
failed to bind to the resin equilibrated with 0.2 M (NH^)
SO^(Figure 3). However, all of the RNA polymerase activity
was bound to the resin. Of the two partially resolved
peaks of A28Q aksorfrani3 material eluted from the resin by
the (NH^^SG,, gradient, shown in more detail in Figure 4,
only the second peak exhibited RNA polymerase activity.
Although the RNA polymerase activity detected with dena
tured DNA was twice that detected with native DNA, the
ratio of specific activities with native and denatured
DNA was constant among the eluted fractions of the second
peak. Attempts to enhance purification by equilibration
of the resin with higher than 0.2 M (NH^^SO^ to reduce
binding of the protein in the first peak (Figure 4) were
unsuccessful. If the salt concentration was increased to
0.21 M, 15 percent of the enzyme activity appeared in the
column flowthrough.


98
be detected; those that can synthesize only RNA and those
that can synthesize both poly(A) and RNA.


product, synthesized with [a-32p]ATP, NTPs, denatured DNA
2+
and Mn resulted in these nearest neighbor frequencies:
CpA, 0.25; ApA, 0.44; GpA, 0.18; UpA, 0.13. The require
ments for incorporation of a labeled nucleotide into acid-
insoluble material and the nearest neighbor frequency
indicate that RNA was synthesized. In the presence of
labeled ATP alone, the ApA frequency of alkaline-hydrolyzed
product was 0.90, and the AMP/adenosine ration was greater
than 100, indicating polyadenylic acid [poly(A)] was syn
thesized. Since UTP was incorporated in the presence but
not in the absence of NTPs, no poly(U) was synthesized,
suggesting that poly(A) may be the only homopolymer
accumulated. Poly (A) synthesis was 90 percent inhibited
by 0.025 mM NTPs, whereas both RNA and poly(A) were syn
thesized with 1.0 mM (or greater) NTPs, suggesting the
mode of poly(A) synthesis differs as a function of the nu
cleotides present, but all products synthesized, were acid-
insoluble after heating (100) for 5 min. in 1 percent SDS,
indicating that product-protein and product-DNA complexes
were not responsible for acid-insolubility. RNA and
poly(A) synthesis was inhibited by 1 yM a-amanitin,
indicating that both products were synthesized by RNA
polymerase II. Poly(A) synthesis was supported by poly(dAdT),
but not by poly(dAT), suggesting a poly(dT) template was
utilized for poly(A) synthesis. Actinomycin D (7 yM)
inhibited RNA synthesis 87 percent on denatured calf thymus
Xll


23
speed and for 15 sec. at high speed. The homogenized
material had the consistency of a thick milkshake. The
homogenized material was immediately filtered through four
layers of cheesecloth, through a layer of miracloth and
into a chilled Erlenmeyer flask. Material retained in the
filters was discarded.
Step 3. High speed centrifugation
The filtered homogenate containing 20 to 40 percent
of the protein present in the shoots and roots was centri
fuged for 60 min. in a Ti50 rotor at 200,000 x g at 0.
The supernatant fraction was decanted into a chilled
graduated cylinder through a layer of miracloth, which re
tained the lipid layer accumulated at the top of the centri
fuge tube. The supernatant fraction contained approximately
50 percent of the protein present in the filtered homogenate.
Step 4. Ammonium sulfate precipitation
To the high speed supernatant an equal volume of
saturated (NH^^SO^ was added slowly with continuous and
gentle stirring in an ice-jacketed beaker. After 30 min.
of stirring the resulting precipitate was collected by
centrifugation (10,000 x g, 10 min., 0) and resuspended
in a minimal volume of Buffer R (approximately 15 ml).


89
Strain et ^1. (47) reported the partial resolution
of two type II maize RNA polymerases during DEAE-cellulose
chromotography; one which preferred native DNA and one
which preferred denatured DNA. The conditions of purifi
cation resulted in very low specific activities, perhaps
reflecting an advanced state of polymerase degeneration.
The possible degeneration of RNA polymerase is supported
by the data of Chambn (39) with the detection of RNA poly
merase protein subunits of decreasing size, designated Bl
and B2. The partial separation of two RNA polymerase
activities by Strain et al. (47) would be consistent with
the presence of one less degenerate RNA polymerase, and
one in an advanced state of degeneration.
Enzyme Characterization
Maize RNA polymerase resembles the type II eukaryotic
RNA polymerases (1). The position of the maize RNA poly
merase in the DEAE-cellulose elution profile at 0.35 M
(NH^^SO^ (Figure 4) was as expected for a type II RNA
polymerase (32). Like the other type II enzyme (32), maize
RNA polymerase can utilize both native and denatured DNA
as a template (Table 4); it requires high salt (Figure 14)
2+ 2+
and either Mg of Mn as metal cofactors (Table 4). The
2+ 24-
strong preference shown for Mn over Mg by the sea urchin,


80
we determined the amount of poly(A) accumulated with dena
tured DNA in a standard reaction mixture as a function of
time. .AMP incorporation in the absence of the NTPs was
not affected by the ribonuclease treatment (Figure 23,
panel B). In data not shown here AMP incorporation in a
standard reaction mixture containing denatured DNA and
4 yg ribonuclease showed no inhibition of incorporation,
consistent with the exclusive synthesis of poly(A). In
the presence of the NTPs, there was a decrease in acid-
insoluble AMP when the product was treated with ribonuclease
(Figure 23, panel A). However, the 10-min. product was
only 10 percent sensitive to treatment whereas the 60-min.
product was 45 percent sensitive. This result suggests
early synthesis of poly(A) and later synthesis of RNA.
Template Requirement
Both calf thymus and maize DNA supported RNA and
poly(A) synthesis (Table 13). In the absence of the NTPs,
where only poly(A) was synthesized, denatured maize and
calf thymus DNA supported equal AMP incorporation. Since
poly(A) synthesis was supported by DNA from both species,
the synthesis of poly(A) was not an artifact of the
heterologous system (calf thymus DNA and maize polymerase).
The lower AMP incorporation in the complete system
observed on denatured maize DNA as compared with denatured
calf DNA reflects the presence of native DNA in the heated
maize preparation; note the low hyperchromicity of the
maize DNA.


5
problems have been solved and the study of in vitro
transcription in eukaryotes is rapidly advancing.
Transcription as a subject may be broken into two
areas of investigation: first, the prokaryotes and their
virases that have served as model systems due to the ease
of acquiring enzyme and defined DNA templates; second, the
eukaryotes that have complex chromosomes and multiple
polymerase.systems producing the intricate specialized
tissues of plants and animals. The bacterial systems will
be illustrated by.the E. coli and B. subtilis systems and
the eukaryotes by the calf thymus and maize systems.
Bacterial Transcription
Significant Concepts
There -are extensive reviews of transcription available
(1,2,4,5,6). I would like to draw from these systems to
illustrate four concepts which have recently been uncovered
in bacterial transcription.
1. RNA polymerase and associated factors have
the ability to initiate and terminate tran
scription at specific sequences on the
chromosome.
. Alteration of the RNA polymerase, either
through viral infection or sporulation,
results in a change in the transcription
specificity of the RNA polymerase.
2


69
consistent with the synthesis of poly(A) before NTP addition.
Enzyme
Enzyme was required for AMP incorporation and
increased as a linear function of the enzyme protein present
(Figure 20). Replacement of denatured DNA with native DNA
caused greater than a 90 percent decrease in AMP incorpora
tion. Therefore, the synthesis of poly(A) was directly
proportional to the enzyme level and the enzyme preferred
denatured DNA.
Product Characterization
The acid-insoluble product synthesized with native
2+ 2+
or denatured DNA with either' Mg or Mn as cofactor
remained insoluble after heating at 100 in 1 percent SDS
for 5 min. (Table 9). As anticipated, if poly (A) longer
than 12 nucleotides was accumulated in the reaction with
denatured DNA, a more acid-insoluble product was synthesized
and it too remained acid-insoluble after treatment.
Nearest neighbor frequency analysis of products
3 2
synthesized from [a- P]ATP in the.absence of the NTPs
resulted in a 0.90 ApA frequency (Table 10), indicating
poly(A) synthesis. Incorporation into CpA, GpA and UpA
accounted for 10 percent of the label incorporated.
The average chain length of the product was estimated
by the ratio of AMP to adenosine after alkaline hydrolysis.
The chromatographed AMP fraction contained 2,200 cpm,


6
3. RNA polymerase may be the site of hormone
action via specific protein factors such
as CAP .(catabolite gene-activator protein)
which are influenced by hormone-controlled
cyclic AMP.
4. Alteration of the RNA polymerase transcription
specificity can result in irreversible
changes in cell phenotype, closely
resembling differentiation in eukaryotes.
These four concepts illustrate the potential importance of
a better understanding of the RNA polymerases in both pro
karyotes and in eukaryotes.
E. Coli RNA Polymerase and Sigma Factor
E. coli RNA polymerase contains four types of subunits:
g' (155-165,000 d), 3 (145-155,000 d), a (85-95,000 d), and
a (39-41,000 d). A fifth subunit, u (10,000 d), is some
times found with the RNA polymerase although it is not
required for RNA polymerase function (4). Together these
subunits are arranged as holenzyme (8'Ba a), which retains
2
the ability to asymmetrically transcribe T4 DNA, or as core
enzyme (g'Ba ), which cannot asymmetrically transcribe T4
2
DNA (11) The presence or absence of sigma (a) determines
the initiation specificity of the RNA polymerase on T4 DNA.
Another factor, rho (p), although not bound to the RNA poly
merase, when present with the enzyme resulted in one type
of specific termination of RNA synthesis (15) Two other
sites also resulted in chain termination. The RNA poly
merase, without rho, also recognized sequences in the DNA
transcribed as UAA (ochre) and UAG (amber) and terminated


97
in opposite directions. In APPENDIX B, a diagramatic
model.^foj the initiation of transcription is presented.
This model includes the initiation of RNA synthesis by
poly(A), the synthesis of RNA on either the same or
opposite DNA strands, and the presence of an RNA poly
merase containing two active sites, each site capable
of binding DNA and of polymerizing nucleotides.
The partially resolved peaks of two maize RNA
polymerase II activities reported by Strain et al. (47)
might reflect a degenerate RNA polymerase that can syn
thesize only RNA, and a less degenerate enzyme that can
synthesize both poly(A) and RNA. The apparent preference
of one RNA polymerase II for denatured DNA may represent
the synthesis of poly(A) by the less degenerate enzyme.
Similarily, the failure to detect homopolymer synthesis
by Stout and Mans (44), may reflect a similar degeneration
of the in vivo RNA polymerase complex. Maize RNA poly
merase purified by the procedure described in METHODS
does retain the ability to synthesize both poly(A). This
rapid purification procedure may be necessary to prevent
the degeneration of the RNA polymerase complex with the
associated decrease in activity, perhaps including the
loss of poly(A) synthesis. Further purification of the
maize RNA polymerase will determine whether such degenera
tion takes place, and whether the two polymerase types can


DNA, but stimulated poly(A) synthesis 28 percent, again
suggesting poly(dT) regions of DNA were utilized as a
template for poly(A) synthesis. Neither RNA nor poly(A)
synthesis was inhibited by cordycepin (0.26 mM) or
rifampicin (50 yg/ml), therefore eliminating the presence
of maize NTP: exotransferase and bacterial RNA polymerase
activity. Since early product (accumulated at a high rate
of AMP incorporation) was resistant to pancreatic ribo-
nuclease, whereas, late product (accumulated at a lower
rate of AMP accumulation) was sensitive to RNase digestion,
poly(A) synthesis apparently preceded RNA synthesis. A
model is presented which requires that poly(A) synthesis
preceda RNA synthesis in the same enzyme-template complex
during the initiation of transcription in eukaryotes.
xiii


22
SEEDLINGS
KERNELS
ROOTS AMD SHOOT
STEP 2 HOMOGENIZE
FILTER BOUND
STEP 3 1 CENTRIFUGE
PELLET
STEP 4 | 50% (NH4)2S04
SUPERNATANT
PELLET
STEP 5 SEPHADEX 650
INCLUDED VOLUME
EXCLUDED VOLUME
tsS
STEP 6 DEAE-CELLULOSE
swsske^
RNA POLYMERASE
Figure 1. PURIFICATION PROCEDURE FOR RNA POLYMERASE


LITERATURE REVIEW
Approaches to Transcription
Guides to Literature
There are several avenues into the literature of
transcription; for current research, the symposia (1,2,3)
and reviews (4,5,6) are best, whereas for a more historical
perspective, there are collected papers (7) and introductory
texts (8,9,10) .
Systems Studied
Essentially every level of life from the smallest
viruses to the largest eukaryotes has had, or is having,
its transcriptional processes studied. The basic enzymology
has been done in bacterial systems, principally Escherichia
coli and Bacillus subtilis and their phages (11,12).
Although RNA polymerase was first detected in a eukaryote
by Weiss in 1960 (13), the bacterial viruses and their
nucleic acids proved most useful in elucidating transcription
in prokaryotic systems (11,14). The major difficulties en
countered in studying the eukaryotic systems were the initial
low activity of the eukaryotic RNA polymerases and the dif
ficulty in getting the enzyme DNA-dependent (15). These
4


17
.The E. coli RNA polymerase had a greater affinity for
denatured DNA than for native DNA (52), perhaps reflecting
a greater binding affinity for the exposed poly(dT) regions
of the denatured DNA.
Importance
From 1361 to 1968, poly(A) synthesis by prokaryotic
RNA polymerase was an unusual artifact of the assay and of
unknown significance. With the discovery of poly(A) se
quences in RNA isolated from numerous eukaryotes; vaccinia
virus cores (53), Hela cells (54,55), mouse sarcoma cells
(56), and avian myeloblastosis virus (57), poly(A) syn
thesis again became of interest. In eukaryotes only a
portion of the DNA-like nuclear RNA is transported to the
cytoplasmic polysomes. In addition, the nuclear RNA is
much larger in size than that found in the cytoplasm (53).
In studying poly(A) synthesis in vaccinia viral cores,
Kates asked, "Could poly(A) sequences in nuclear RNA
play a role in either the cleavage of RNA into smaller
pieces or in .the selective transport of certain species
to the cytoplasm?" (53, p. 752). To answer the question
of the role of poly(A), two additional questions should
first be answered. Is the poly(A) covalently attached to
the RNA, and if so, where? If attached to RNA, what
enzyme catalyzed the synthesis of the poly(A) sequence?
Finding some poly(A) attached to RNA does not imply that


60
was RNA. The relatively high ApA frequency derived from
product synthesized with denatured DNA suggested the
presence of poly(A) regions in the product.
Summary
The maize RNA polymerase required added DNA, the
four nucleosidetriphosphates; ATP, GTP, CTP and UTP, and
2+ 2+
a metal, either Mg or Mn for activity. The maximum
activities with native and denatured DNA were equal when
2+
Mg was a cofactor. The maximum activity with denatured
. 2+
DNA was twice that with native DNA if Mn was the metal
cofactor. Enzyme activity was salt-dependent and pro
portional to the amount of enzyme protein added. The
substrate reached saturation above 1.0 mM and the reaction
continued for at least 90 min. The product with denatured
DNA had a high ApA nearest neighbor frequency.
Polyadenylic Acid Synthesis
The RNA synthesis data indicated that an additional
activity might be present in the RNA polymerase reaction.
These indications were: 1) With denatured DNA there was
significant incorporation of AMP in the absence of the NTPs.
2) There was a doubling in activity with denatured DNA
2+ 2+
when Mn replaced Mg but with native DNA there was little
change. 3) There appeared to be a biphasic rate of AMP


77
_ g
indicated 50 percent inhibition at 1 x 10 M (Figure 21).
Since a-amanitin is specific for the type II eukaryotic
KNA polymerase (32), this inhibition of activity indi
cated that the maize enzyme was a type II RNA polymerase
and that it catalyzed both RNA and.poly(A) synthesis (69).
Actinomycin D inhibited AMP incorporation in the
presence of the NTPs (Table 12). However, in the absence
of the NTPs, AMP incorporation was stimulated. Even at
very high actinomycin D concentrations (70 yM), there was
only a slight inhibition of poly(A) synthesis. Therefore,
the sensitivity of the RNA polymerase to actinomycin D was
dependent upon the presence of the NTPs. In the presence
of the NTPs, AMP incorporation was inhibited 50 percent
at 2 yM actinomycin D and was inhibited 95 percent at 50 yM
(Figure 22) .
The inhibition by a-amanitin and the resistance to
cordycepin and rifampicin excluded the presence of two
potential enzyme contaminants (Table 12). The maize NTP:
exotransferase is resistant to a-amanitin and sensitive
to cordycepin while bacterial RNA polymerases are resistant
to a-amanitin and sensitive to rifampicin. Therefore the
presence of NTP: exotransferase activity from maize tis
sue and bacterial RNA polymerase activity from contaminat
ing bacteria were ruled out.
Utilizing the resistance of purine-purine
phosphodiester linkages to pancreatic ribonuclease (54),


65
TABLE 6
AMP AND UMP INCORPORATION BY RNA POLYMERASE
A standard reaction mixture containing 0.37 A2gQ denatured
DNA, 0.4 ymoles MnCl2/ either 0.10 ymoles labeled ATP or
0.05 ymoles labeled UTP and 4 yg enzyme.
Labeled Nucleotide Incorporated
System (pmoles)
14 14
C-AMP C-UMP
Complete
381
Complete plus NTPs
913
22
458C
aTotal radioactive nucleotide incorporated per reaction
mixture.
NTPs
contained
0.25
ymoles
each:
GTP,
CTP
and
UTP .
NTPs
contained
0.25
ymoles
each:
GTP,
CTP
and
ATP.


18
all the poly(A) was attached, or remained attached.
Knowing the time and location of poly(A) synthesis and the
enzymes responsible would contribute significantly to an
understanding of the physiological function of poly(A)
synthesis.
Location in Vivo
Poly(A) can be isolated from the HnRNA or from the
rapidly labeled RNA isolated from polyribosomes (54,55,56).
Edmonds (54) indicated the data was consistent with the
idea that every HnRNA contained at least one poly(A) se
quence. More than one mode of poly(A) synthesis was
implicated since cordycepin (3-deoxyadenosine) suppressed
the labeling of mRNA found on Hela ribosomes while not
effecting the labeling of nuclear RNA (58). This suggested
there may be two enzymatic activities that synthesized
poly(A), one sensitive to cordycepin that synthesized
poly(A) and one insensitive to cordycepin that synthesized
HnRNA containing poly(A) sequences. Lim and Cannelakis'
(59) results with haemoglobin mRNA indicated that, at most,
it could contain 70 polypurine residues of 70 percent AMP.
Furthermore, this polypurine sequence was not at the 3'
end of the haemoglobin mRNA, since the 3' end contained
only 7 or 8 AMP residues before the first pyrimidine (60).
Therefore, the poly (A) must be at the 5' end or inside the
RNA chain, if there at all.


84
TABLE 14
RNA POLYMERASE ACTIVITY WITH SYNTHETIC TEMPLATES
Enzyme activity assayed in a standard reaction mixture con
taining 0=5 ymoles MgCl2, 0.037 &260 DNA' either 0.1 ymole
labeled ATP or 0.05 nmole labeled UTP and 4 yg RNA poly
merase.
Nucleic Acid Substrate Incorporation (pmoles^1
AMP UMP
Poly(dAT)
14
C-ATP
13
-
Poly(dAT)
14
C-ATP
+
UTP
1,090
-
Poly(dAdT)
14
C-ATP
104
-
Poly(dAdT)
14
C-ATP
+
UTP
211
-
Poly(dAdT)
14
C-UTP
-
4
Poly(dAdT)
14
C-UTP
+
ATP
-
40
Poly(dAdT)
14
C-UTP
+
Id
ATP (a-aman)
-
0.2
aTotal radioactive nucleotide incorporated per reaction
mixture.
^a-amanitin added to 20 yg/ml.


39
bands on the gel run with native protein may reflect greater
sensitivity of band detection since the sample analyzed con
tained 'twice as much protein and the gel was stained with
aniline blue black rather than coomassie brilliant blue.
Assuming that the distance migrated by the denatured protein
components in SDS-polyacrylamide gels was a function of
their molecular weights, then the pair of bands migrating
21 and 23 mm (equivalent to the migration of BSA) were
.about 65,000 to 75,000 d. Therefore, the other bands
characteristic of the RNA polymerase preparations corre
sponded to polypeptides of higher molecular weight.
Enzyme Stability
Storage temperature and freeze-thaw
Maize RNA polymerase collected in glass vials and
immediately stored in liquid nitrogen lost no activity
after 3 months. The enzyme was stable for 19 days at -16,
but lost all activity at -17 (Table 2, part A). The
addition of glycerol to 20 percent by volume to fresh
enzyme preparations did not stabilize the polymerase
enough to make storage at -17 practical. However, in 20
percent glycerol the enzyme did retain 50 percent activity
for 17 h at 4 (Table 2, part B). The enzyme stored in
liquid nitrogen was stable to repeated freeze-thaw. The
loss of activity observed may reflect decay accumulated
during the time the enzyme was held on ice preceding assay
(Table 2, part C).


76
TABLE 12
INHIBITOR SENSITIVITY OF MAIZE RNA POLYMERASE
A standard reaction mixture contained 0.037 A
DNA, 0.5 ymoles MnCl2, 0.05 ymoles ATP, 5 yg
hibitors as indicated added at zero times.
2gg denatured
enzyme and in-
Inhibitor Concentration
Percent
of Control
Plus NTPsa
Less NTPs^
a-amanitin 0.1 yM
6
7
1.0 yM
1
2
Actinomycin D 7.0 yM
14
128
70.0 yM
1
87
Cordycepin 0.13 mM
100
101
0.26 mM
-
100
Rifampicin 50.0 yg/ml
100
110
a336 pmoles AMP incorporated equaled
100%.
^138 pmoles AMP incorporated equaled
100%.


75
conclusion was indirect, since the enzyme preparation was not
homogeneous. The data presented thus far in support of one
enzyme catalyzing both syntheses includes: 1) Both activ
ities were eluted simultaneously from DEAE-cellulose by
an (NH^^SO^ gradient with a constant RNA and poly (A) activity
ratio in the active fractions. 2) Both activities required
DNA for AMP incorporation. 3) Both activities had the
2+ 2+
same Mg and Mn cofactor optima. 4) The for ATP was
the same with native and denatured DNA. Additional com
parisons of RNA and poly(A) synthesis were made to further
associate the accumulation of both products with one
enzyme. These included inhibitor activities, nuclease
sensitivity of products as a function of time and template
specificity.
Effect of Inhibitors
Three types of inhibitors of RNA polymerase were
investigated, those that bind specifically to the enzyme
protein such as a-amanitin and rifampicin; those that
bind to the template such as actinomycin D; and those
that compete with the substrate such as cordycepin. The
inhibitors were tested both in the presence and absence
of the NTPs, therefore measuring the sensitivity of both
RNA and poly(A) syntheses.
Low concentrations of a-amanitin inhibited both RNA
and poly(A) activity equally (Table 12). A titration of
RNA polymerase activity with increasing levels of a-amanitin


Assayed in a standard reaction mixture containing 0.37 A denatured DNA and 0.5 ymole
MnCl2; panel A with NTPs, panel B without NTPs, and 5 yg^RNA polymerase. At the times
indicated reaction mixtures were removed and either precipitated in 10% trichloroacetic
acid or incubated with 0.2 yg pancreatic ribonuclease (see METHODS). RNase treated samples
indicated by open circles. \ 1 y
co
i


100
REFERENCES
- 1. .Cold Spring Harb. Symp. Quant. Biol. (1970). 35.
2. Silvestri, L. (ed.). Lepetit Colloquia on Biol. and
Med. (1969). American Elsevier Pub. Co., 52 Vander
bilt Ave., New York, N.Y.
3. Hanly, E. W. (ed.). RNA in Development, The Park City
Inter. Symp. on Prob. in Bio. (1969), Univ. of Utah
Press, Salt Lake City, Utah.
4. Burgess, R. R. (1971). Ann. Rev. Biochem., 40:711.
5. Von Hippel, P. H., and J. D. McChee (1972). Ann. Rev.
Biochem., 41:231
6. Losick, R. (1972). Ann. Rev. Biochem., 41:409.
7. Zubay, G. L. (ed.). Papers in' Biochemical Genetics,
1968). Holt, Rinehart and Winston, Inc., New York,
N. Y.
8. Watson, J. D. Molecular Biology of the. Gene., 2nd ed.
(1970). W. A. Benjamin, Inc., New York, N.Y.
9. Mahler, H. R. and Cordes, E. H. Biological Chemistry
(1966). Harper and Row, New York, N. Y.
10. Lehninger, A. L. Biochemistry (1971). Worth Publishers,
Inc., New York, N. Y.
11. Burgess, R. R., A. A. Travers, J. J. Dunn and E. K. F.
Bautz (1969). Nature, 221:43.
12. Losick, R. and A. L. Sonenshein (1969). Nature, 224:35.
13. Weiss, S. (1960). Proc. Nat. Acad. Sci., 46:1020.
14.Shorenstein, R. G. and R. Losick (1972). Fed. Proc.,
31:472. abs.

LO
i1
Chamberlin,
Biol., 35
M.
: 851.
(1970) .
Cold Spring Harb. Symp
16.
Roberts,
J.
W.
(1969) .
Nature, 224:1168.
17.
Travers,
A.
A.
(1970) .
Nature, 225:1009.


37
<-)
A H B
Figure 5. POLYACRYLAMIDE GEL ELECTROPHORESIS OF
NATIVE RNA POLYMERASE
Samples were run on 5.2% polyacrylamide gels at pH 8.8
(see METHODS). Gel A contained 40 yg peak RNA poly
merase (eluted at 235 ml, Figure 4). Gel B contained
20 yg BSA.


the polymerase utilized different sequences in the DNA
but not necessarily different DNA molecules for RNA and
poly(A) synthesis.


54
0 4 8
I
ATP
Figure 13. LINEWEAVER-BURKE PLOTS OF ATP
TITRATIONS
Assayed- in a standard reaction mixture con
taining 0.5 limles MnCl2, 0.37 A2gg DNA, either
native (solid circles) or denatured (solid
triangles), and 4 yg RNA polymerase. The ATP
concentration was mM with the K at 0.125 mM.
The was 100 moles AMP/mg/2U min. for de-
nature§XDNA and 50 nmoles AMP/mg/20 min. for
native DNA.


36
Polyacrylamide Gel Electrophoresis
Antaliquot of the proteins showing the highest RNA
polymerate specific activity (eluted from DEAE-cellulose
at 235 ml in Figure 4) was examined by electrophoresis on
5.2 percent polyacrylamide gel. Several components were
visible, with more than 10 bands staining with aniline
blue black (Figure 5). All the protein staining material
migrated into the gel, toward the anode at pH 8.8. There
fore, the proteins are acidic and none were excluded by
the 5.2 percent polyacrylamide gel. The slowest migrating
and darkest staining band, however, may represent an a gregate of some of the more rapidly moving components. The
intensity of this major band was approximately equal to
that of the BSA standard (20 ug) and, therefore, may account
for 50 percent of the added protein. If an aliquot of the
same protein preparation was denatured with 0.1 M 2-mercap-
toethanol and 0.1 percent SDS, then subjected to electro
phoresis on 10 percent polyacrylamide-0.1 percent SDS gels,
the number of visible bands was reduced to 5 (Figure 6).
Two distinct bands, migrating 13 and 14 mm into the gel, a
faint band at 19 mm ^nd a still fainter pair of thin bands
at 21 and 23 mm were characteristic of all the purified
maize RNA polymerase preparations. The decrease in detect
able bands on SDSpolyacrylamide as compared to the 5.2 per
cent polyacrylamide gels may reflect disaggregation of the
denatured proteins. Alternatively, the presence of more


67
TABLE 7
INHIBITION OF POLYADENYLIC ACID SYNTHESIS
BY NUCLEOTIDES
RNA polymerase activity measured in a standard reaction
TT!'ixt.ure containing 0.037 A260 denatured UNA, 0.05 ymoles
.MnC.12 / 5 yg enzyme, and nucleotides as indicated.
Additional Nucleotides AMP Incorporated3 (pmoles)
GTP, CTP, UTP (0.25 ymoles each) 619
None 210
UTP (0.3 ymoles) 40
GTP (0.3 ymoles) 31
CTP (0.3 ymoles) 43
UTP (0.03 ymoles) 50
a
Total pmoles AMP incorporated per reaction mixture.


101
REFERENCES (continued)
.18.
Seifert, W., D. Rabussay and W.
'Lett., 16:175.
Sillig
(1970) FEBS
T9.
Goff, C. G. and K. Weber (1970). Cold
Symp. Quant. Biol., 35:101.
Spring Harb.
20.
Emmer, M., B. DeCrombrugghe, I.
(1970). Proc. Nat. Acad. Sci
Pastan and R. L. Perlman
., 66:480.
21.
Riggs, A. D. and S. Bourgeois
9.-A84.
(1969).
Biophys. J.,
22.
Jost, J. and H. V. Rickenberg
chem., 40:471.
(1971) .
Ann. Rev. Bio-
23.
Aronson, A. J. (1969). J. Mol
. Biol.,
11:576.
24.
Hussey, C., R. Losick and A. L.
J. Mol. Biol., 57:59.
Sonenshein (1971).
25.Zubay, G. D. Schwartz and J. Beckwith (1970). Proc.
Nat. Acad. Sci., 66:104.
26. Keller, W. and R. Goor (1970). Cold Spring Harb. Symp.
Quant. Biol., 35:671.
27. Stout, E. R. and R. J. Mans (1967). Biochem. Biophys.
Acta., 134:327.
28.Jacob, S. T., E. M. Sajdel and H. N. Munro (1968).
Biochem. Biophys. Acta., 157:421.
29. Seifart, K. H. and C. E. Sekeris (1967). Hoppe-
Seylers Z_. Physiol. Chem., 348 :1555 .
30. Cunningham, D. D., and D. F. Steiner (1967). Biochem.
Biophys. Acta., 145:834.
31. Roeder, R. G., and W. J. Rutter (1970). Proc. Nat.
Acad. Sci., 65:675.
32.Blatti, S. P., C. J. Ingles, T. J. Lindel, P. W. Morris,
R. F. Weaver, F. Weinberg, and W. J. Rutter (1970).
Cold Spring Harb. Symp. Quant. Biol., 35:649.
Hearst, J. E. and M. Botchan (1970). Ann. Rev. Biochem
39:151.
33.


INTRODUCTION
Macromolecular nucleic acid metabolism depends upon
two classes of enzymes: the polymerases which assemble
the polymers from the nucleoside or deoxynucleoside tri
phosphates, and the nucleases which break down the polymers.
Among the polymerases there are four types which are known
to utilize a nucleic acid as a template to synthesize a
new polymer. These are DNA-dependent DNA polymerase
(EC 2.7.7.7), DNA-dependent RNA polymerase (EC 2.7.7.6),
RNA-dependent RNA polymerase and RNA-dependent DNA. poly
merase. Of primary importance in phenotypic expression of
genetic information are the DNA-dependent RNA polymerases.
These enzymes are responsible for the synthesis of messenger
RNA,the template for sequencing amino acids, and for the
synthesis of ribosomal and transfer RNAs, needed for trans
lation of the messenger RNA into protein. Control of the
synthesis of these RNAs seems to be primarily at the level
of initiation of transcription.
Transcription may be studied in vivo or in vitro.
The in vitro approach to transcription requires a purified
RNA polymerase. Once the enzyme is purified, specific
properties can ten be determined, such as the assay
1


24
Step 5. Salt equilibration on sephadex
Equilibration in Buffer E was accomplished by passage
erf -the "'resuspended precipitate through a sephadex G50
column(2.5 x 11 cm) equilibrated with Buffer E. The ex
cluded material was diluted to 25 ml with Buffer E for
absorption to DEAE-cellulose.
Step 6. DEAE-cellulose chromotography
The excluded volume eluted from sephadex G50
chromotography was loaded (1 ml/min.) onto a DEAE-cellulose
(see materials) column (2.5 i.d. x 11 cm) equilibrated with
Buffer E. The loaded column was washed with 60 ml of
- -,vt
Buffer E and the flow rate was decreased to 0.25 ml/min.
A 60 ml (NH^)2S0^ gradient (0.20 to 1.0 M) was begun
immediately after passage of the 60 ml of Buffer E.
Column eluates were monitored and recorded by a continuous
flow ultraviolet-monitoring system (Gilford spectrophoto
meter and a Honeywell recorder, Figure 2). Eluted fractions
were collected directly from the flowcell into glass vials
(2.5 ml/vial) and frozen in liquid nitrogen.
Polymerase Assay
RNA polymerase was assayed in a 0.10 ml standard
reaction mixture containing: 10 ymoles Tris-Hcl, pH 7.6 @
25; 0.25 ymoles each UTP, CTP, GTP, (sodium salts);
0.10 ymoles [8--^C]ATP (specific activity 1.7 to 4.5 yC/ymole) ;
1.0 ymole MgC^; 1*0 ymole 2-mercaptoethanol; 8 ymoles


48
similar but the maximum level of AMP incorporation differed.
Metal
For maize RNA polymerase activity there was an
absolute 'requirement for a metal ion cofactor satisfied
2+ 2 +
with, either Mg or Mn (Table 4) There was a broad
2-§-
Mg optimum concentration from 10 'to 14 mM, centered at
12 mM, and independent of the nativity of the DNA (Figure 9).
Routinely, however, 10 to 25 percent more activity was
observed with denatured DNA than with native DNA. The
2+
maximum AMP incorporation occurred with 5 mM Mn either
with native or denatured DNA (Figure 10). With denatured
DNA, the RNA polymerase activity was twice that with native
2+
DNA at all Mn levels. As is characteristic of polymerases,
2+
the Mn optimum concentration was sharper than that of
,, 2+
Mg
Substrate
With native DNA, 96 percent of the AMP incorporation
required the presence of the NTPs (Table 4). The RNA poly
merase reaction was saturated with the pooled NTPs above
1.0 mM for each of the three nucleoside triphosphates;
GTP, CTP and UTP and no nucleotide inhibition was detected
with a 5-fold excess (Figure 11). The standard reaction
was 2.5 mM with each NTP; thus the assay of polymerase was
not limited with respect to unlabeled nucleosidetriphosphates
The RNA polymerase reaction mixture was saturated


7
RNA chains (15) .
In response to infection by coiiphage T4, E. coli
RNA polymerase transcribed early T4 RNA. After production
of the gene 55 protein, translated from the early RNA, the
synthesis of delayed early RNA was specifically initiated
(16). Travers (17) isolated the protein and called it a
T4 sigma-like factor which caused specific asymmetric
initiation of T4 DNA at the site of delayed early RNA. This
isolation of a factor which directed delayed early RNA
synthesis demonstrated for the first time that RNA poly
merase could acquire a new initiation specificity (16,17).
One minute after infection by phage T4, the host RNA poly
merase no longer synthesized early T4 RNA (18). This
change was apparently caused by alteration of the a sub
unit of the host RNA polymerase through adenylation with
5'AMP (19). Therefore, viral infection initiated changes
in the transcription machinery which caused a specific
alteration of the initiation sites for RNA synthesis.
These changes included alteration of the existing host RNA
polymerase and the synthesis of a viral protein to replace
the host sigma factor.
E. Coli RNA Polymerase and CAP
RNA polymerase control in coli illustrates the
possible importance of transcription as a mechanism for
hormone action. Cyclic AMP, together with CAP, was required
for maximum expression of the lactose and other inducible


AMP INCORPORATED pmoles
58
0 50 100
TIME min
Figure 16. RATE OF AMP INCORPORATION AS A
FUNCTION OF TIME
Assayed in a standard reaction mixture (7X) each
containing 1.0 ymole MgC^/ 0.37 A260 na'^:'-ve
and 18 ug RNA polymerase. The reactions were
incubated at either 30 (solid circles) or 37
(solid triangles) and 0.1 ml aliquots removed at
the times indicated.


PURIFICATION OF A EUKARYOTIC
RNA POLYMERASE II THAT SYNTHESIZES
POLYADENYLIC ACID
By
ROBERT HENRY BENSON
A DISSERTATION PRESENTED TO THE GRADUATE
COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
IS 72


KEY TO ABBREVIATIONS
A
260
ATP
AMP
BSA
CAP
cpm
CTP
d
DMSO
dpm
DEAE
DNA
GTP
g
h
HrRNA
NTPs
POPOP
PPO
poly(A)
poly(dAT)
poly(U)
poly (cLAdT)
absorbancy at 260 run
adenosine triphosphate
adenosine monophosphate
bovine serum albumin
catabolite gene-activator protein
counts per minute
cytidine triphosphate
dalton
dimethyl sulfoxide
disintegrations per minute
diethylaminoethane
deoxyribonucleic acid
guanosine triphosphate
gravity
hour
heterogeneous nuclear ribonucleic acid
GTP, CTP, UTP
1.4-bis-[2-(4-methyl-5-phenyloxazolyl)]-
benzene
2.5-diphenyloxazole
polyadenylic acid
alternating copolymers of deoxyadenylic
and deoxythymidylic acid
polyuridylic acid
homopolymers of deoxyadenylic and
deoxythymidylic acid-
ix


16
Polyadenylic Acid
Enzymatic Synthesis
A eukaryotic polyadenylic acid polymerase was first
discovered in calf thymus by Edmonds and Abrams in 1962
(48). Its activity was inhibited by the other nucleotide
triphosphates, it required magnesium and it was particulate,
perhaps bound to the nuclear membrane. Its product was
almost pure poly(A), except for about 1 percent of the
adenylate residues which were joined to cytidylate resi
dues. The enzyme could not be freed of endogenous RNA.
Others have purified poly(A) polymerases from rat liver
(49) and from maize (50). The maize poly(A) polymerase
adds poly(A) chains to the 3' hydroxyl of primer nucleic
acids, either RNA or DNA (51).
Poly(A) synthesis has also been studied in prokaryotic
systems. E. coli RNA polymerase, with denatured calf thymus
DNA and only ATP as substrate, will synthesize poly(A)
sequences using poly(dT) regions of the DNA as template
(46). Reiteritive transcription of poly(dT) regions, each
greater than 5 nucleotidyl residues long, resulted in the
synthesis of long poly(A) chains through a repeated
utilization of the poly(dT) template by an unknown mechanism
Poly(A) synthesis required denatured DNA and was inhibited
by the addition of the other nucleoside triphosphates.
The polymerase would not lengthen added poly(A) primers (46)


APPENDIX B
A Model for Polyadenylic Acid Initiation
of Transcription
Enzyme Structure
The RNA polymerase consists of a core enzyme with
paired subunits (a2, 82)* Additional factors or subunits
may confer template specificity or additional activities
upon this core polymerase. Each pair of subunits (a,8)
of the core enzyme contains the ability to bind DNA and
to catalyze ENA synthesis, therefore, creating the
potential for two active sites per RNA polymerase complex
(Figure 26) .
Enzyme Activity
Properties of Site 1
Site 1 synthesizes RNA and will bind Randomly on
2+ 2+
denatured DNA. It will utilize either Mg or Mn and
it will incorporate all four NTPs. It is sensitive to
cs-amanitin and actinomycin D. It will actively catalyze
RNA for more than 90 min. in vitro.
109


88
(Table 1). Stout and Mans (27) had concentrated the dilute
Tris-HCl eluted enzyme by salt precipitation. Because the
(KrH^) 2^0^ eluted enzyme was concentrated, the salt precipita
tion was obviated, and the 70 to 90 percent loss of activity
such precipitation, caused, was avoided. In addition to
the removal of most of the contaminating proteins, most, if
not all, nucleic acids were removed. The A,
i/A-
of the
260' 280
material added to the column was 10, whereas the A26c/A28Q
of the eluted fractions containing polymerase was 1.5 to
2.0. Removal of nucleic acids during DEAE-cellulose
chromotography apparently decreased the stability of the
'iEEA. polymerase. The polymerase was stable to salt precipi
tation before, but not after, chromotography. Addition of
DNA, to the eluted fractions containing the polymerase,
prior to precipitation with (NH^)2SO^ improved the recovery
of polymerase activity (Table 3), supporting the premise
that the polymerase requires nucleic acid for stability.
The recovery of active enzyme free of nucleic acid
may, therefore, result from the short time between elution
from DEAE-cellulose and storage in liquid nitrogen. During
the absence of nucleic acid, the RNA polymerase complex
may either aggregate or disassociate, but in either case
it becomes inactive. This hypothesis was supported by the
polyacrylamide gel electrophoresis data (Figure 5). The
presence of one dark band and multiple smaller bands might
represent subunits which have aggregated and/or disassociated
from the polymerase complex.


43
denatured protein exhibited those bands characteristic of
eukaryotic RHA polymerases. The enzyme wat stable after
'freezing and-storage at -7 6. Precipitation of the purified
enzyme with (NH^)2S04 was facilitated by DNA. Further
resolution of the proteins in the RNA polymerase preparation
was thwarted by the instability of the enzyme at 4 and by
the lack of recovery of active enzyme following salt
precipitation.
RNA Synthesis
Assay Requirements
Enzyme purified by the procedure described in METHODS
exhibited the expected requirements for RNA synthesis.
DNA, metal and all four ribonucleosidetriphosphates were
required for incorporation of labeled AMP into acid-insoluble
v
material (Table 4). Denatured DNA was utilized as well as
native calf thymus DNA, and maize DNA (not shown here) sup-
2+
ported comparable activity. Mn satisfied the metal re-
2+ 2+
quirement as well as Mg in fact, better than Mg when
denatured DNA was provided as the nucleic acid component.
*
Incorporation of AMP in the absence of NTPs was significant,
especially when assayed in the presence of denatured DNA.
All of the radioactivity detected in acid-insoluble material
on filter paper disks was dependent upon the addition of
enzyme protein.


15
were 14-16 S, which corresponded to the distribution of
the denatured DNA template (44) This distribution was
that expected for DNA-RNA hybrids. As expected, upon
heating the complexes disaggrated and all nucleic acids
were at the top of the gradient (4-6 S).
The RNA polymerase purified by the method of Stout
and Mans was identified as a type II nucleoplasmic RNA
polymerase by its sensitivity to a-amanitin (45) This
type II RNA polymerase did not synthesize homopolymers
such as poly(A) with denatured DNA, as many bacterial
RNA polymerases did (46), for with denatured DNA "the
formation of a homopolymer was not detected with the maize
polymerase" (44, p. 752), nor was it detected with any
other eukaryotic RNA polymerase.
A type I maize RNA polymerase was reported by Strain
et al. (47) Maize leaves were used as crude material and
carried through DEAE-cellulose chromotography. The type I
RNA polymerase eluted at 0.08 M (NH^^SO^ (47). Strain,
et al. also detected two overlapping peaks of activity in
the type II RNA polymerase region. One peak preferred
native DNA, the other preferred denatured DNA as measured
by total AMP incorporated (47) The metal requirements of
the leaf RNA polymerase indicated a preference for magnesium
over manganese, with optimums at 2 5 mM for magnesium and
8 mM for manganese (47).


DISCUSSION
Enzyme Preparation
Studies of both product characterization and the
initiation of transcription required an enzyme of a much
higher specific activity than the soluble maize RNA poly
merase purified by Stout and Mans (27). In addition, the
net yield of active polymerase had to be increased and the
purification procedure made more convenient and rapid for
.effective experimental progress. These goals were achieved.
The RNA polymerase specific activity was increased 10-fold,
the net yield was more than doubled, and the time required
for each preparation was reduced from 7 days to 10 hours.
These improvements resulted from 4 major alterations in the
procedure of Stout and Mans (27), including changes in:
1. the storage of the starting material; 2. the homogeniza
tion procedure; 3. the salt equilibration of the soluble
proteins; 4. the DEAE-cellulose chromotography procedure.
Previously (27), adequate grain was germinated under
running water and/after 5 days, the seedlings were harvested
just before each enzyme preparation. In contrast, after
large scale germination and harvest (1 to 3 kg) followed
by storage of the seedlings at -76 (Reveo freezer) in
86


53
Figure 12. ATP TITRATION OF RNA POLYMERASE
Assayed in a standard reaction mixture containing
1.0 ymole MgC^/ 0.37 nati-ve NA, 5 yg RNA
polymerase andzATP as indicated.


14
.using plant chromatin or crude salt fractionated supernatants
(40,41,42). ENA polymerase activity was first detected in
the soluble fraction of a French pressure cell extract in
1964 by Mans and Novell! (43) The RNA polymerase in
this extract was further purified by DEAE-cellulose chrorao-
tography using a linear Tris-HCl gradient (Stout and Mans
1967) (27). The average specific activity eluted was 4.06
nmoles AMP/mg at 10 min. This eluted RNA polymerase would
utilize either native or denatured DNA as a template
equally well, although at low DNA levels (10 ug DNA/ml) dena
tured DNA was eight times as effective as native DNA (44).
Denatured calf thymus DNA was more efficient as a template
than denatured maize DNA; however, the calf thymus DNA had
a much greater hyperchromicity than maize DNA, 17 percent
vs 8.3 percent (44). The RNA polymerase required all four
nucleoside triphosphates, a bivalent metal ion, and DNA to
14
incorporate [8- C]ATP into acid-insoluble material (27).
The metal could be either magnesium (25 mM) or manganese
(5mM) If [ct-32p]UTP or [ct-32p]ATP were used as labeled
substrate, the nearest neighbor frequency indicated RNA
containing all four nucleosidemonophosphates had been syn
thesized (27) The reaction was inhibited by actinomycin D,
pyrophosphate and DNase (43). The product synthesized on
native DNA was greater than 90 percent digested by pancreatic
ribonuclease, while on denatured DNA this decreased to 73
percent (27). On sucrose density gradients the products
1


103
REFERENCES (continued)
51. Mans, R. J. (1971). Biochem Biophys. Res. Comm.,
45:980. "
52. Hurwitz, J., J. J. Furth, M. Anders and A. Evans
(1962). J. Biol. Chem., 237:3752.
53.Kates, J. (1970). Cold Spring Harb. Symp. Quant.
Biol., 35:743. -
54. Edmonds, M., M. H. Vaughan, Jr., and H. Kakazato (1971)
Proc. Nat. Acad. Sci. 68:1336.
55.
Darnell, J. E., R.
Proc. Nat. Acad.
Wall
Sci. ,
and R. J. Tushinski (1971).
£8:1321.
56.
Lee, S. Y., J. Mendecki,
Proc. Nat. Acad. Sci.,
and G. Brawerman
68:1331.
(1971).
57.
Green, M., and M.
£9:791.
Cartas
(1972). Proc.
Nat. Acad. Sci.
58.
Penman, S. H, M..
Roshas
h and S. Perlman
(1970), Proc.
Nat. Acad. Sci., 67:1878.
59. Lim, Y., and E. S. Cannelakis (1970). Nature, 227:710.
60. Burr, H. and J.. B. Lingrel (1971). Nature N. B.,
233:41.
61. Heaust, J. E., and M. Botchan (1970). Ann. Rev. Bio
chem. 39:177.
62. Lowry, O. H., N. J. Rosenbough, A. L. Farr and R. J.
Randall (1951). J. Biol. Chem., 193:265.
63. Hulbert, R. B., in S. P. Colowick and N. 0. Kaplan
(ed.)f Methods in Enzymology, 3_:21a.
64. Davis, B. J. (1964). New York Academy Science, 121:404
65. Weber, K., and M. Osborn (1969). J. Biol. Chem., 244:
4406.
66. Peterson, E. A., and H. A. Sober, in S. P. Colowick and
N. 0. Kaplan (ed.) Methods in Enzymology, £:3.
67. Burgess, R. R. (1969). J. Biol. Chem., 244:6168.
68.Gellespie, D., and. S. Speigelman (1965). J. Mol. Biol.
12:820.


82
TABLE 13
COMPARISON OF CALF THYMUS AND MAIZE DNA AS
TEMPLATES FOR RNA POLYMERASE
Assayed in a standard reaction mixture containing 0.5 ymoles
MnC^, 0.037 A^gg DNA anc^ ^.4 ^9 P^Y617356
DNA
£
AMP Incorporated (pmoles)
Complete Less NTPs
Native Calf Thymus
219
29
Native Maize
260
46
Denatured Calf Thymus*3
400
160
. c
Denatured Maize
300
163
aTotal pmoles incorporated
per reaction
mixture.
uHyperchromicity 26%.
cHyperchromicity 11%.


3
the initial synthetic activity of the RNA polymerase
and to minimize the influence of any other enzymatic
activities present. In addition, experimental manipu
lation of the enzyme during purification should be kept
minimal, to reduce the probability of enzyme degeneration
and, therefore, to preserve in vivo transcriptional
activity. The experimental approach in this study was
to purify the RNA polymerase from maize seedlings, to
determine its requirements for product accumulation and
to identify the products. RNA polymerase was purified
by centrifugation, (NH^)2SC>4 precipitation and ion ex
change column chromotography in that order. From its
assay requirements and inhibitor sensitivities it was
identified as SNA polymerase type II. In addition to its
known catalysis of RNA synthesis, this eukaryotic RNA
polymerase catalyes polyadenylic acid synthesis. The
polymerase products were characterized by sensitivity to
heat, SDS and pancreatic ribonuclease, and by nearest
neighbor frequency analysis. The possible involvement of
poly(A) synthesis as a mechanism of initiation of tran
scription is suggested and discussed.


CONCLUSION
Maize DNA-dependent RNA polyirierase II synthesizes
both RNA and polyadenylic acid utilizing a DNA template.
The evidence is consistent with a model where polyadenylic
acid synthesis precedes the synthesis of RNA in the same
enzyme-template complex during the initiation of
transcription.
99


LIST OF FIGURES
1. PURIFICATION PROCEDURE FOR RNA POLYMERASE 22
2. DEAE-CELLULOSE GRADIENT ELUTION ASSEMBLE ..... 25
3. DEAE-CELLULOSE ELUTION PROFILE' 34
4. RNA POLYMERASE ACTIVITY IN DEAE-CELLULOSE
FRACTIONS 35
5. POLYACRYLAMIDE GEL ELECTROPHORESIS OF NATIVE RNA
POLYMERASE 37
6. POLYACRYLAMIDE GEL ELECTROPHORESIS OF DENATURED
RNA POLYMERASE .38
7. DNA TITRATIONS WITH MAGNESIUM AS COFACTOR 46
8. DNA TITRATIONS WITH MANGANESE AS COFACTOR 47
9. MAGNESIUM TITRATION WITH NATIVE AND DENATURED DNA. 49
10. MANGANESE TITRATION WITH NATIVE AND DENATURED DNA. 50
11. NTP TITRATION OF. RNA POLYMERASE. 51
12. ATP TITRATION OF RNA POLYMERASE 53
13. LINEWEAVER-BURKE PLOTS OF ATP TITRATIONS 54
14. AMMONIUM SULFATE TITRATION OF RNA POLYMERASE ... 55
15. RNA POLYMERASE ACTIVITY AS A FUNCTION OF ENZYME
CONCENTRATION. . 57
16. RATE OF AMP INCORPORATION AS A FUNCTION OF TIME. 58
17. DNA TITRATIONS WITH MAGNESIUM IN THE ABSENCE OF
NTPs 62
18. DNA TITRATIONS WITH MANGANESE IN THE ABSENCE OF
NTPS 63
vii


93
remain available for reiterative poly(A) synthesis. With
NTP addition, reiterative transcription would cease, RNA
synthesis would begin, and the enzyme would encounter an
actinomycin D-guanosine complex and be inhibited. There
fore, both the NTP inhibition and actinomycin D inhibition
data are consistent with poly(A) and RNA synthesis occurring
on adjacent regions of the same DNA template.
Poly(A) and RNA are synthesized by the maize.RNA
polymerase II, and may be attached to one another. Maize
RNA polymerase synthesized RNA with native and with de
natured DNA, but the ApA frequency went from 30 percent
with native DNA (27) to 44 percent with denatured DNA
(Table 5). The increase in ApA frequency was not neces
sarily a change in the base composition of the RNA syn
thesized, but may have resulted from the simultaneous
synthesis of poly(A) and RNA on the denatured DNA template.
The early product synthesized in a complete reaction
mixture containing denatured DNA was resistant to
pancreatic ribonuclease, while late product was more
sensitive (Figure 23). This suggested the initial product
contained poly(A) and the subsequent product contained
RNA. Early poly(A) synthesis was also suggested by the
kinetics of AMP incorporation: a rapid initial rate in
which apparently all the nucleotides incorporated were
labeled; and a subsequent lower rate in which approximately
30 percent of the incorporated nucleotides were labeled


29
swollexu.a:ri£l .^jillibrated in 0.05 M Tris-HCl, pH 7.6 at 25,
and;^rfcor^di''ai(i:4^?,^Bovine serum albumin, 5X crystallized,
was..:£:rom PentexSEiochemicals. The nucleoside triphosphates
GTP, CTP, UTP -and. ATP were purchased as sodium salts from
Schwarz/Mann, including [8-l^C]ATP, [2--^C]UTP and [a-32p]
ATP. Calf thymus DNA was purchased from Schwartz/Mann, and
pancreatic ribonuclease, chromatographically pure, was
purchased from Worthington Biochemicals. Actinomycin D
was purchased..tfr:em Schwarz/Mann and cordycepin, grade C,
was purchased from Sigma. Alpha-amanitin was a gift of
Dr. T. Weiland and rifampicin was a gift from Gruppo-
Lepetit S.P.A. Research Laboratory. Zea mays L., WF9 x
Bear 38, waxy, was purchased from the Bear Hybrid Seed Co.
Reagents
Calf thymus DNA was dissolved in 0.1 x SSC at 37
ml and stored in 0.2 ml aliquots at -17 (67). An aliquot
was thawed for use before each assay. Denatured DNA was
prepared by the dilution of a freshly thawed aliquot of
DNA in 0.1 x SSC (1:1 v/v) into a sealed vial, 5 min.
immersion in boiling water, followed by quick chilling^in
iced-water (average hyperchromicity 24%). All the &260
measurements in the tables and figures represent the
absorbancy before denaturation.
Stock solutions of 1.0 M Tris-HCl, pH 8.0 or pH 7.6
at 25, 1.0 M MgC^, 14.7 M 2-mercaptoethanol were used to
make the following buffers: Buffer H, 0.25 M sucrose,


92
Poly (A) and SNA are probably synthesized on the
same template. Addition of the NTPs increased AMP and
UMP incorporation equally (Table 6) reflecting RNA
synthesis. The increased AMP incorporation occurred above
the level of homopolymer synthesis measured when the NTPs
were absent. However, if very low. levels of NTPs were
added, poly (A) synthesis was inhibited (Figure 19). This
inhibition was identical to the NTP inhibition of E. coli
PJSTA polymerase during poly (A) synthesis by reiterative
transcription (46). The E. coli RNA polymerase was in
hibited when the enzyme began transcribing RNA with an
insufficient concentration of NTPs to support measurable
RNA synthesis. This resulted in a net inhibition of
AMP incorporation. Since delayed addition of low level
NTPs inhibited maize poly (A) and RNA synthesis, the syn
thesis of poly(A) arid SNA probably occurred on the same
DNA molecule. Utilization of the same DNA molecule for
poly(A) and RNA synthesis was further supported by the
actinomycin D resistance of poly(A) synthesis. In the
absence of the NTPs, poly(A) synthesis was refractory
to actinomycin D, but when NTPs were added, all incorpo
ration was inhibited by the actinomycin D (Table 12).
Since actinomycin D preferentially binds guanosine and
inhibits RNA synthesis by impeding the progress of the
enzyme along the DNA template (70), the poly(dT)-rich
regions of the DNA, unable to bind actinomycin D, would


87
aluminum packets, each enzyme preparation required only the
removal of a weighed packet of tissue from the freezer just
before homogenization. The specific activities of the
homogenates from freshly harvested tissue and from tissue
stored at -76 were identical.
In the original procedure (27), the shoots and roots
were pulverized under liquid nitrogen and then, in small
batches, passed through a French pressure cell. This was
replaced by rapid homogenization, in one batch, in a
Waring blender. During homogenization, the enzyme was
protected from oxidation (foaming) with 50 mM 2-mercapto-
ethanol. The enzyme specific activity of the blender
treated material (Table 1) was identical to material from
the French pressure cell (27).
The removal of (NH^^SO^ from the salt-precipitated
enzyme fraction by a 4 h dialysis against Buffer R (27),
was replaced by a 20 min. gel filtration procedure. The
Sephadex G50 was equilibrated and the proteins eluted in
the excluded volume with 0.2 M (NH^)2SO^. At this salt
concentration all RNA polymerase was bound to the DEAE-
cellulose column, but only a small fraction of the total
protein was bound (illustrated by A2gQ in Figure 3).
Previously (27) RNA polymerase was eluted with a
shallow Tris-HCl gradient (500 ml, 0.05 to 1.0 M). This was
replaced by a steep (NH^)2SO^ gradient (60 ml, 0.2 to 1.0 M),
that eluted concentrated, highly active, RNA polymerase


AMP INCORPORATED pmoies
Figure 7. DNA TITRATIONS WITH MAGNESIUM AS COFACTOR
Assayed in a standard reaction mixture containing 1.0 ymole MgCl2
4 yg RNA polymerase and DNA as indicated; native (solid circles) ,
denatured (solid triangles) and native-denatured mixture (open circles) .


41
Salt precipitation
-.w jtotasr.a.t^Tempt to concentrate the RNA polymerase eluted
sfrccra- DEX2-:eelUiXose, solid (NH^^SO^ was added to 8 0 per
cent saturation to the pooled fractions. Only 10 to 15 per
cent of the eluted activity was recovered in the precipitated
protein. To facilitate precipitation, calf thymus DNA was
added in varying amounts to the pooled fractions just before
salt addition. Addition of DNA resulted in an increase in
precipitated activity, roughly porportional to the amount
of DNA added (Table 3). Low recovery of polymerase in
precipitates from a solution with high DNA levels (300 pg/
ml) suggested ..formation of a DNA-protein complex soluble in
high salt. Neither polymerase activity in the supernatants
nor protein in the precipitates was determined, therefore
inactivation of polymerase and specific activity were not
assayed per se.
Summary
DNA-dependent RNA polymerase was purified 1,000-fold
from an homogenate of maize shoots and roots. Following
DEAE-cellulose chromotography the enzyme specific activity
was greater than 100 nmoles AMP incorporated/mg enzyme at
20 min. The recovery was greater than 50 percent of the
activity detected in the high speed supernatant. Poly
acrylamide gel electrophoresis of both native and SDS-
denatured proteins indicated the RNA polymerase preparation
still contained several polypeptides. Nevertheless the


PERCENT ACTIVITY
79
Figure 22. ACTINOMYCIN D TITRATION OF RNA POLYMERASE
Assayed in a standard reaction mixture containing 1.0 ymole
MgCl2, 0.37 A2g0 native DNA, 18 yg RNA polymerase and
actinomycin D as indicated. Inhibitor was added just prior
to addition of enzyme to the reaction mixture.


68
TABLE 8
EFFECT OF DELAYED ADDITION OF NTPs ON
POLYADENYLIC ACID SYNTHESIS
A standard reaction mixture containing 0.5 ymoles MnC1^>
0.037 &260 denatured DNA, 5 yg enzyme and NTPs as indicated
were \ised. Delayed addition of the NTPs was at times in
dicated during a standard 20 min. incubation. NTPs con
tained GTP, CTP and UTP.
NTPs Added
AMP Incorporated5
pmoles %
None 160 40
0.25 yiQoles each, 0 min. 400 100
0.0025 pmoles each,
0 min. 52 13
1 min. 60 15
5 min. 88 22
aTotal pmoles incorporated per reaction mixture.


71
TABLE 9
EFFECT OF HEATING AND SDS ON
RNA POLYMERASE PRODUCT
Standard reaction mixture with 0.37 A2gQ DNA, and either
1.0 ymoles MgCl2 or 0.4 ymoles MnCl2 as indicated. Heating
was for 5 min. at 100. SDS was 1% where indicated. Each
assay contained 6.5 yg enzyme.
Components in Reaction Mixture
AMP Incorporated
(pmoles)*
Template
Metal
Control
Heated
Heated in SDS
Native
Mg
660
610
625
Native
Mn
627
632
630
Denatured
Mg
805
800
840
Denatured
Mn
1160
1160
1190
Total radioactive nucleotide incorporated per reaction
mixture.


LIST OF FIGURES (continued)
19. NTP TITRATION WITH DENATURED DNA 66
20. POLYADENYLIC ACID SYNTHESIS AS A FUNCTION OF
ENZYME CONCENTRATION. 70
21. ALPHA-AMANITIN TITRATION OF RNA POLYMERASE. ... 78
22. ACTINOMYCIN D TITRATION OF RNA POLYMERASE .... 79
23. RIBONUCLEASE SENSITIVITY OF DENATURED DNA-DEPENDENT
PRODUCTS. 81
24. AMP INCORPORATION AS A FUNCTION OF ATP SPECIFIC
ACTIVITY 106
25. RATE OF AMF INCORPORATION AS A FUNCTION OF ATP
CONCENTRATION 108
26. A MODEL FOR POLYADENYLIC ACID INITIATION OF
TRANSCRIPTION 110
viii


I certify that I have read this study and that in
ray opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Rusty Jjl^ans, Chairman
Prq^'essor of Biochemistry
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Daniel Billen
Professor of Radiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
John W. Cramer
dissociate Professor of
Pharmacology and Therapeutics
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree, of Doctor of Philosophy.
George E. Gifford
Professor of Immunology and
Medical Microbiology