Mitochondrial RNA synthesis and ribonucleotide incorporation studies in Euglena gracilis

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Mitochondrial RNA synthesis and ribonucleotide incorporation studies in Euglena gracilis
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xii, 109 leaves : ill. ; 28 cm.
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Brown, George Erwin, 1939-
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Euglena gracilis   ( lcsh )
RNA   ( lcsh )
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Microbiology thesis Ph. D
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Thesis--University of Florida.
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Bibliography: leaves 98-107.
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by George Erwin Brown.
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Typescript.
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Vita.

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Full Text













MITOCHONDRIAL RNA SYNTHESIS AND RIBONUCLEOTIDE INCORPORATION STUDIES
IN Euglena gracilis










by








GEORGE ERWIN BROWN


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
1976


























This dissertation is dedicated to my wife, Carolyn M. Brown, my parents,
Mr. and Mrs. Marshall V. Brown, and my children, George E. Brown and
Erika N. Brown, for their endless encouragement and personal sacrifices
during the time I spent in graduate school.

















ACKNOWLEDGEMENTS


The author wishes to express his gratitude to the members of his

doctoral committee for their advice, guidance and assistance in the

preparation of this dissertation. Special acknowledgements of thanks

are given to the author's major professor and advisor, Dr. J. F. Preston,

for providing the laboratory facilities and supplies for conducting the

research reported here. The laboratory techniques learned in his labora-

tory have already proved to be rewarding for the author in his pursuit

of other research projects. The use of Euglena gracilis as a test organism

for studying organelle development originated with Dr. Preston. He con-

tributed much of his normal off duty time offering advice and suggestions

toward the completion of this study.

The author also thanks Dr. R. F. Mans for the personal interest he

took in the author's research and academic development. The advice and

guidance he provided proved to be valuable in pursuing the study of

nucleotide incorporations. The consultations held with Dr. Mans provided

the stimulation to complete this research rather than to terminate it.

In addition the knowledge obtained in the "Rusty Mans Molecular Journal

Club" was most rewarding in terms of the author's academic development.

Expressions of gratitude are also in line for Dr. L. O. Engram for

his encouragements when things looked bad and for his valuable suggestions

that led to the completion of this research. Dr. Engram provided chemicals

and laboratory facilities for conducting several experiments.










Dr. E. M. Hoffmann will be long remembered as the author's first

graduate instructor and for inspiring the author's interest in microbiology.

The author also thanks him for his assistance in the preparation of this

dissertation and for providing laboratory equipment for conducting this

research.

The author wishes to express his gratitude to Dr. T. W. O'Brien for

serving as the mitochondria expert and for providing advice and guidance

in the initial phase of this research. The consultations held with

Dr. O'Brien provided the author with the assistance and encouragements for

completing this dissertation.

















TABLE OF CONTENTS


Page

ACKNOWLEDGMENTS . . . iii

LIST OF TABLES . . . vi

LIST OF FIGURES . . vii

KEY TO ABBREVIATIONS . . .. viii

ABSTRACT . .... . .. x

INTRODUCTION . . . 1

LITERATURE REVIEW . ... .. .. 8

MATERIALS AND METHODS . . .. 24

RESULTS . . . 34

DISCUSSION ......... . .. 91

CONCLUSIONS. .. . . ... 97

REFERENCES . . .. 98

BIOGRAPHICAL SKETCH . . .. 108

















LIST OF TABLES


Page
1. DISTRIBUTION OF SUCCINIC DEHYDROGENASE ACTIVITY................... 41

2. NUCLEOTIDE INCORPORATION BY ISOLATED MITOCHONDRIA ................. 42

3. INCORPORATION OF RIBONUCLEOSIDE TRIPHOSPHATES BY ISOLATED
MITOCHONDRIA ..................................................... 46

4. THE DEPENDENCY OF THE MITOCHONDRIAL ACTIVITY INCORPORATING LABEL
FROM 3H-CTP UPON ADDED RIBONUCLEOTIDES AND DNA.................... 47

5. DISTRIBUTION OF 3H-CMP INCORPORATING ACTIVITY..................... 48

6. INHIBITION OF 3H-CMP INCORPORATION................................. 50

7. CHARACTERIZATION OF PRODUCT.............................. .... 51

8. PHOSPHOLIPID EXTRACTION OF THE 3H-CMP LABELED PRODUCT............ 60

9. A COMPARISON OF THE INCORPORATION OF H-UTP AND H-CTP BY
ISOLATED MITOCHONDRIA PURIFIED BY TREATMENT WITH DNASE I..........71

10. RNA POLYMERASE ACTIVITY SOLUBILIZED FROM MITOCHONDRIA .............73

11. SUCCINIC DEHYDROGENASE ACTIVITY IN THE MITOCHONDRIAL SUPERNA-
TANTS AND PELLETS AFTER SOLUBILIZATION TREATMENT................. 75

12. THE EFFECT OF AMMONIUM SULFATE PRECIPITATION ON MITOCHONDRIAL
RNA POLYMERASE ACTIVITY ......................................... 77

13. A COMPARISON OF TRITIUM INCORPORATION FROM H-UTP AND H-CTP
BY THE ACTIVITY IN THE DEAE-SEPHADEX A-25 COLUMN FRACTION 3...... 82

14. THE DEPENDENCY OF THE DEAE COLUMN FRACTION 38 INCORPORATION OF
3H-UMP UPON ADDED RIBONUCLEOSIDE TRIPHOSPHATES AND DNA........... 83

15. INHIBITORS OF 3H-UMP INCORPORATION BY THE DEAE COLUMN FRACTION
38........................... ...................... ................ 84

16. TEMPLATE SPECIFICITY OF THE DEAE COLUMN FRACTION 38.............. 90

















LIST OF FIGURES
Page
1. SCHEMA FOR THE ISOLATION OF MITOCHONDRIA.......................... 35

2. DENSITY EQUILIBRIA ANALYSIS OF MITOCHONDRIAL DNA.................. 38

3. RIBONUCLEOSIDE TRIPHOSPHATES INCORPORATION BY ISOLATED
MITOCHONDRIA..... ............................. ................... 43

4. SUCROSE GRADIENT ZONAL CENTRIFUGATION............................ 53

5. BIOGEL P-4 COLUMN CHROMATOGRAPHY................................. 55

6. ULTRA VIOLET ABSORBANCY SPECTRA.................................. 58

7. THIN LAYER CHROMATOGRAPHY OF THE 3H-CMP LABELED PRODUCTS ELUTED
FROM THE BIOGEL P-4 COLUMN....................................... 61

8. THIN LAYER CHROMATOGRAPHY OF THE PRODUCTS OF THE ENZYMATIC
CLEAVAGE OF THE 3H-CMP LABELED MATERIAL ELUTED FROM THE
P-4 COLUMN........................................................ 64

9. THE MIGRATION OF THE ENZYMATIC CLEAVAGE PRODUCTS CONTAINING
TRITIUM LABEL ON PEI CELLULOSE................................... 66

10. DENSITY EQUILIBRIA ANALYSIS OF DNA FROM DNASED MITOCHONDRIA........ 69

11. PROCEDURE FOR THE PARTIAL PURIFICATION OF MITOCHONDRIA RNA
POLYMERASE........................................................ 78

12. ION EXCHANGE CHROMATOGRAPHY ON DEAE-SEPHADEX A-25................. 80

13. DEPENDENCE OF THE ACTIVITY OF THE DEAE FRACTION 38 ON THE
CONCENTRATION OF Mn+ ............................................. 86

14. DEPENDENCE OF THE ACTIVITY OF THE DEAE FRACTION 38 ON THE
CONCENTRATION OF Mg+ ............................................. 88

















KEY TO ABBREVIATIONS


A260

A280

AMP

ATP

ATPase

BGP- 1


BGP- 2


BSA

CAP

CDP-diglyceride

CMP

CpC

cpm

CTP

dCTP

cyclic AMP

DEAE

DNA

DNase

dpm

DTT


absorbancy at 260 nm

absorbancy at 280 nm

adenosine monophosphate

adenosine triphosphate

adenosine triphosphatase

the first peak of tridium labeled material
eluted from Biogel P-4 column

the second peak of tridium labeled material
eluted from Biogel P-4 column

bovine serum albumin

catabolite gene-activator protein

cytidine diphosphate diglyceride

cytidine monophosphate

cytidylyl (3' 5') cytidine

counts per minute

cytidine triphosphate

deoxycytidine triphosphate

adenosine 3' : 5'-cyclic monophosphoric acid

diethylaminoethane

deoxyribonucleic acid

deoxyribonuclease (EC 3.1.4.5)

disintegrations per minute

dithiothreitol


viii










ethylenediamine tetraacetate

gravity

guanosine monophosphate

guanosine triphosphate

hour

radioactively labeled with tridium


I

NTPs

poly d(AT)


poly (C)

POP

POPOP

RNA

Hn RNA

m RNA

r RNA

t RNA

RNase

S

SDS

SSC

SVP

TCA

tris

UMP

UTP

UV


Iodine

ATP, CTP, GTP, UTP (minus the labeled substrate)

alternating copolymers of deoxyadenylic and
deoxythymidylic acid

poly cytidylic acid

2, 5 diphenyloxazole

1, 4 di-[2-(5-phenyloxazolyl)]-benzene

ribonucleic acid

heterogeneous nuclear ribonucleic acid

messenger ribonucleic acid

ribosomal ribonucleic acid

transfer ribonucleic acid

ribonuclease (EC 2.7.7.16)

sedimentation coefficient

sodium dodecyl sulfate

standard saline citrate

snake venom phosphodiesterase

trichloroacetic acid

tris hydroxymethyll amino) methane

uridine monophosphate

uridine triphosphate

ultra violet


EDTA



GMP

GTP

hr
3H










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



MITOCHONDRIAL RNA SYNTHESIS AND RIBONUCLEOTIDE
INCORPORATION STUDIES IN
Euglena gracilis



By



GEORGE ERWIN BROWN

August, 1976




Chairman: James F. Preston, Ph.D
Major Department: Microbiology



Two experimental approaches were utilized to study the in vitro

mitochondrial RNA synthesis in Euglena gracilis. First the RNA syn-

thesis activity was examined in isolated and highly purified mitochondria

of Euglena gracilis strain z streptomycin bleached aplastic mutants. The

mitochondria obtained after isopynic centrifugation in sodium diatrizoate

gradients yield mitochondrial DNA (p=1.691 g/cm3) and little nuclear DNA

(p=1.707 g/cm ). The incorporation of label from the H-ribonucleoside

triphosphates into acid insoluble products showed no dependence upon the

presence of the other ribonucleotides or that of added DNA. The preferred

substrate was 3H-CTP from which label was incorporated into acid insoluble

products at a rate 100 times greater than the other labeled ribonucleotides.

This activity incorporates CMP from CTP into acid insoluble products










which contain phosphodiester bonds by a pyrophosphorolysis reaction. The

activity was inhibited by pyrophosphate and actinomycin D at 100 mg/ml but

not by inorganic phosphate, a-amanitin, rifampicin or low concentrations of

actinomycin D (50 mg/ml). The products were sensitive to snake venom phos-

phodiesterase and alkaline hydrolysis but not pancreatic ribonuclease. The

products were isolated by phenol extraction followed by ethanol precipita-

tion of the aqueous phase. Two classes of molecules with mean molecular

weights of 800 and 680 respectively were isolated. They were found to have

absorbancy spectra identical to that of CpC and were resistant to pancreatic

ribonuclease digestion. However, snake venom phosphodiesterase cleaved the

products into 5'-CMP, the expected cleavage product of CpC or poly C.

Mitochondria, purified by either isopycnic centrifugation or by treat-

ment with pancreatic deoxyribonuclease, contained an associated activity

which incorporated label from 3H-UTP into acid insoluble products which

were sensitive to pancreatic ribonuclease digestion. This activity was

DNA dependent in the DNase mitochondria which was in contrast to that

observed for sodium diatrizoate purified mitochondria.

The second approach was to study the activity of solubilized and

partially purified mitochondrial RNA polymerase. An enzyme purification

procedure is described utilizing a solution of Triton X-100 detergent and

KC1, centrifugation, ammonium sulfate precipitation, and DEAE-Sephadex A-25

chromotography that resulted in a partially purified enzyme with a specific

activity of 0.3 nmoles UTP/mg/10 min at 370. The incorporation of UMP into

acid insoluble material required DNA the four ribonucleoside triphosphates:

GTP, CTP, UTP, ATP, and metal, either Mn- or Mg The product of the

reaction was sensitive to pancreatic ribonuclease digestion. The mito-









chondrial enzyme is distinctly different from the nuclear RNA polymerase II

with respect to chromatographic behavior and antibiotic sensitivity. It

eluted from the DEAE-Sephadex column in a single peak between 0.32 M and

0.37 M ammonium sulfate in TGMED buffer, was stimulated by low concentration
of the metals Mn and was insensitive to a-amanitin and rifampicin.
of the metals Mn and was insensitive to a-amanitin and rifampicin.







/ ^
















INTRODUCTION


Informational macromolecules are those through which genetic in-

formation actually flows. Genetic information may be defined as that

primarily required to assemble a protein needed to perpetuate biological

order, but the definition applies also to the information for ribonucleic

acids (RNAs) which are not translated into protein. Deoxyribonucleic

acids (DNA) functions as the repository for information or coding se-

quences ultimately destined to appear either in RNA or protein, and in

noncoding sequences which can act as signals for such things as initiation

and termination of the biosynthesis of a RNA chain. Within the cell the

informational sequences are transmitted on which specific proteins are

synthesized.

The DNA dependent RNA polymerases (E. C. 2.7.7.6.) are the key

enzymes implicated in the first step of genetic information flow from DNA

to protein. The term DNA dependent RNA polymerases is used to designate

enzymic activities which, using DNA templates, catalyze the sequential

assembly of the four ribonucleoside triphosphates into RNA molecules.

The synthetic reaction requires the presence of a DNA or a polydeosy-

ribonucleotide template and a divalent metal ion. The main features of

the transcription reaction consist of the enzyme locating and binding to

specific sites on the DNA, initiating a RNA chain de novo, extending the

chain by moving along the DNA using only one strand as a template to

direct the polymerization of the four ribonucleoside triphosphates into











a complementary RNA molecule, and finally, in some cases, termination of

the chain with the release of the enzyme from the DNA template. RNA

polymerases are responsible for the synthesis of ribosomal RNA's, transfer

RNA's and messenger RNA's, all of which are needed for translation of the

mRNA template into proteins.

Substantial data have been accumulated on the regulation of RNA

polymerases in prokaryotes, where a single enzyme is responsible for the

synthesis of all types of cellular RNA, but comparatively little is

known in the case of eukaryotes, where the situation is more complex.

Within the last few years, it has been demonstrated that eukaryotic cells

contain several types of RNA polymerases, which differ in their locali-

zations, structures, and functions.

Nuclei contain several different RNA polymerases. Nuclear RNA

polymerase I is localized in the nucleolus (1). It is primarily con-

cerned with the transcription of genes specifying the larger (45s)

ribosomal precursor RNA. Nuclear RNA polymerase II is localized in the

nucleoplasm and presumably transcribes messenger RNA. It is inhibited

by a-amanitin (2,3) an octapeptide produced by several species of the

genus Amanita (4). In several organisms a third (5,6) or even a fourth

(7,8) nuclear RNA polymerase activity was found. A separate RNA poly-

merase III, which is distinct from forms I and II, has been shown to

transcribe (4s) t-RNA and 5s RNA (9,10). In mammals this enzyme is

slightly sensitive to a-amanitin, being 1,000 fold more resisistant than

RNA polymerase II.

RNA polymerase activity has also been found localized in mito-

chondria and chloroplasts. The mitochondrial enzymes exhibit properties











which clearly distinguish them from the nuclear RNA polymerases. The

mitochondrial enzyme is insensitive to a-amanitin and although some

functional differences have been described in mitochondrial RNA poly-

merase, there is general agreement that the molecular weight of the


enzyme, consisting of a single polypeptide chain, is about 60,000

daltons (11-15).


Control of RNA synthesis for regulating cell
growth, development, and differentiation

If considerations were based purely on energy requirements, it

would be reasonable to exclude DNA replication and mRNA translation as

primary levels where gene expression is controlled. Several reasons may

be given for excluding changes in the kinds or numbers of genes and post-

transcriptional modification as steps at which regulation generally takes

place. Nuclei of cells in different stages of development in the same

individual appear to be genetically identical and changes in the number of

gene copies from which mRNA is transcribed have not been observed. Like-

wise, there is no good evidence that changes in the kinds of proteins

synthesized in development can be attributed to controls at the trans-

lational level and in addition eukaryotic cells have no mechanism for

excluding the translation of messages characteristic of other cell

types (16). It seems that translational controls are used to make quanti-

tative adjustments to a pattern of protein synthesis determined primarily

by the synthesis of new messages.

It can be concluded by elimination that transcription must be the

level at which gene expression is primarily controlled. In bacterial

systems, there are two well-characterized examples of programmed trans-










scription: bacteriophage development and bacterial sporulation. These

processes are similar in some ways to differentiation. Therefore, under-

standing the control of transcription in these processes may provide some

clues to the regulation of transcription in eukaryotic systems. Studies

of transcriptive regulation of phage X show how the expression of specific

genes is regulated by other gene products (17). In both T4 bacteriophage

development and Bacillus subtilis sporulation, significant changes that

are coordinated with development occur in the RNA polymerase molecule

and associated proteins (18,19,20). In B. subtilis there is evidence

suggesting that these changes may be related to the onset of sporulation.

The primary need for regulating transcription is that, by turning

it on and off, protein synthesis is controlled at the origin. In both

prokaryotic and eukaryotic organisms regulation must enable cells to

cope with fluctuation in their immediate environment. In the prokaryote

most of the genes come into operation sooner or later during each cell

cycle, but this is not true of many genes in the higher eukaryote where

differentiation has resulted in a strategy of regulation unlike that in

the prokaryote. By contrast the lower unicellular eukaryote has a re-

gulatory resemblance to the prokaryote. Evidence that gene transcription

is controlled in eukaryotic development is provided by the Balbiani rings

of polytene chromosomes. The ring is characteristically expanded and

active in RNA synthesis or unpuffed and inactive in transcription at

different stages of development (19). Also, agents such as actinomycin

which inhibit RNA synthesis but which have little immediate effect on

translation arrest eukaryotic development within a short time. These

effects suggest an essential role for the regulation of transcription in

development.










Regulation of transcription by alteration
in molecular properties of RNA polymerase

The regulation of transcription may be affected by control of any

of the components involved in the RNA synthesis: the template, the poly-

merase, associated factors and other environmental conditions. However,

the multiplicity and different intranuclear localization of DNA-dependent

RNA polymerases have suggested that gene expression in eukaryotic cells

is regulated, in part, by distinct RNA polymerases with different template

specificities. The quantity and quality of RNA synthesis can depend

either on the concentration of RNA polymerase or on the level of activity

of the enzyme. In growing E. coli cells, there is a pool of excess RNA

polymerase (21) although the polymerase subunits seem to be synthesized

at the same rate as other cellular proteins (22). Therefore, the regula-

tion of growth depends upon modulation of the RNA polymerase activity

rather than upon the concentration of the enzyme. Although the rRNA

genes account for only about 0.4% of the genome (23), rRNA accounts for

40% of the total RNA synthesized in growing cells and is therefore

preferentially synthesized (24).

On the other hand, bacteriophage infection results in the inhibition

of host cell transcription presumably by the destruction or alteration of

the host RNA polymerase (25,26). Similar effects on host RNA synthesis

have been found in viral infections in mammalian cells (27,28). This

may be analogous to terminal differentiation where the loss of nuclear

function is involved (29)


Regulation of transcription and control of
organelle development

An understanding of the mechanisms by which a eukaryotic cell

controls organelle development should give valuable insight into many










similar cellular control processes such as those involved in differentia-

tion, morphogenesis and tumor production. Since RNA polymerase has been

implicated in controlling development in prokaryotic systems (18,19,25,

30,31) it could be a major controlling factor in Euglena gracilis organelle

development. Therefore, the research in this laboratory focuses upon the

RNA polymerases found in Euglena, and upon their role in organelle de-

velopment. Euglena is an excellent organism to study transcription in

organelles since it contains both chloroplasts and mitochondria. The

organism is a unicellular flagellate with 10 to 12 chloroplast per cell,

numerous mitochondria, and a polyploid nucleus. Euglena can be grown

phototrophically, heterotrophically or mixotrophically. These growth

conditions will alter the development of the organelles and therefore

offer a useful system for studying organelle development. Phototrophic

conditions are restrictive for photosynthetic function, but the other

two growth conditions are permissive. In cells grown phototrophically,

mitochondrial development is repressed, while the chloroplasts function

to provide the bulk of the cell's energy. Cells grown mixotrophically

derive energy from functional chloroplasts and mitochondria. However,

wild type cells can be grown in the dark as etiolated heterotrophs

where the proplastids, which have stopped forming chlorophyll and

chloroplast membrane, exist as undeveloped proplastids which are

capable of differentiating into functional mature chloroplasts upon

exposure to light. The heterotrophic dark grown cells primarily use

mitochondrial oxidative phosphorylation as a source of energy. It has

been shown (32) that cells grown heterotrophically have large active

mitochondria, and that exposure to light, which starts plastid develop-










ment, results in a decrease in mitochondrial density and function (33).

Mutants that completely lack plastids (aplastidic) have been produced by

treatment with streptomycin, heat, or ultraviolet light (34,35). These

permanently bleached cells are perfectly viable as long as they are

grown on a carbon source which can be respired. This makes it feasible

to distinguish processes controlling mitochondrial development from those

controlling chloroplast development.

This dissertation concerns itself with studies of mitochondrial RNA

synthesis in Euglena gracilis and is directed towards elucidating the

components of the transcriptional process, in particular, characterizing

the mitochondrial DNA dependent RNA polymerase with a view toward under-

standing the control of transcription. The RNA synthesizing activity of

isolated mitochondria has been studied and the DNA dependent RNA polymerase

has been solubilized and partially purified. The experimental approach

in this study was to develop a method for preparing highly purified mito-

chondria, to study the incorporation of labeled ribonucleoside triphosphate

precursors into RNA by isolated mitochondria and to identify the labeled

products. The mitochondrial DNA-directed RNA polymerase was partially

purified by ion exchange chromatography. The enzyme activity was charac-

terized to determine its requirements for product synthesis and to compare

it to nuclear activities with respect to these requirements.

















LITERATURE REVEIW

Guides to the Literature


In recent years, several review articles or symposia on RNA poly-

merases from prokaryotes (20,36-46) and eukaryotes (47-52), on the

regulatory elements (53,54) and on transcription (55-59), have appeared.

There are several introductory texts (60-62) and collected papers (63-66)

which may be consulted for a more historical background discussion.


General Mechanisms of RNA Synthesis

The synthesis of RNA from DNA is mediated by a DNA dependent RNA

polymerase that uses ribonucleoside triphosphates as precursors. In

general the reaction involves binding of enzyme to DNA template and

the asymmetric transcription of the DNA. RNA synthesis appears to proceed

in the following steps: template binding, chain initiation, chain elon-

gation and termination. The RNA polymerase binds to specific initiation

sites (promoter sites) on the DNA in a specific reaction in which the

strands of the DNA are opened over a short local region. Chain initiation

involves the binding of two ribonucleotide triphosphates to the RNA

polymerase followed by elimination of inorganic pyrophosphate to form a

dinucleotide tetraphosphate. The initial nucleotide retains its triphos-

phate while each added ribonucleotide triphosphate has its two terminal

phosphates cleaved as pyrophosphate. The direction of elongation is

from 3' hydroxy to 5' phosphate in the DNA, the RNA being made 5' to 3'










in the antiparallel direction. The initial product is complementary to

the region of the DNA employed as template and the RNA may be modified

post transcriptionally.


Prokaryotic Transcriptions

Background

There are several reviews of prokaryotic transcription available

(35,36,56,57) which may be consulted for discussions of aspects that

will not be considered here. The basic transcriptional studies have

been carried out in bacterial systems with and without phage infection

and these studies have served as a model system for understanding the

transcriptional process and its control. Only the properties of the

bacterial RNA polymerase and the regulation of bacterial RNA synthesis

will be reviewed here.


Prokaryotic RNA polymerase

The bacterial RNA polymerases are large molecules (molecular

weights between 400,000 and 500,000) and have complex subunit structures.

Two enzymatically active forms of the RNA polymerase are currently known:

the holoenzyme and the core enzyme. The holoenzyme contains the following

polypeptide chain subunits: one beta prime (3') subunit; one beta (8)

subunit; two alpha (a) subunits; and either one sigma (o) subunit or one

sigma prime (o') subunit. The subunits have the molecular weights of

approximately 160,000; 155,000; 90,000; and 40,000 respectively. Two

forms of the holoenzyme with the structures a2BB'a(67) and a2BG''(68)

have been found in Escherichia coli. The core enzyme lacks a sigma

subunit. Therefore, the holoenzyme can be separated into two functional

parts: a core enzyme, which is able to synthesize RNA but lacks the










ability to initiate such synthesis specifically; and a sigma subunit,

which acts catalytically to allow the efficient initiation of RNA at

specific promoter sites. Bacterial RNA polymerases from different

species appear to be closely related in subunit structure but show dif-

ferences in the sizes of the subunits (42). Some preparations of the

E. coli enzyme contain a minor component w (69,70) which has a molecular

weight of about 10,000 and is present in the stoichiometry of two w per

holoenzyme. The subunits of the holoenzyme are functional subunits

since it has been demonstrated that these subunits are essential for the

reconstitution of enzymic activity when the enzyme is reformed from

separated subunits (71,72). The w subunit is not required for recon-

stitution of RNA polymerase holoenzyme activity.

All transcription in E. coli is sensitive to inhibitors of the

purified enzyme, such as rifampicin and streptolydigin (73,74), which

block RNA synthesis through interaction with the B subunit of the

enzyme (75). Thus, all RNA synthesis appears to depend on an enzyme

complex in which the B subunit is functional. The beta subunit is also

altered in enzymes from mutants resistant to rifampicin (71) or

streptolydigin (76-78).


General mechanism of prokaryotic transcription

Transcription is a process involving a series of different bio-

chemical events. The holoenzyme is able to recognize and to bind to

specific regions on the DNA template (the promoters) and to undergo a

conformational change, catalyzing the addition of substrate ribonucleo-

side triphosphates to the binding site (79), with the subsequent for-

mation of the first internucleotide linkage of ATP or GTP with a second










ribonucleoside triphosphate. Inorganic pyrophosphate is eliminated,

resulting in the formation of a dinucleotide tetraphosphate of the general

structure pppPupX. The sigma subunit appears to be released (80,81) and

the core enzyme moves along the DNA template synthesizing the RNA chain

by adding ribonucleoside monophosphates to the 3' hydroxy terminus of the

nascent RNA chain from ribonucleoside triphosphate substrates. When the

RNA polymerase encounters a specific termination site on the DNA template,

RNA chain growth is terminated and the nascent RNA chain and the RNA

polymerase are released from the template. A termination protein factor,

rho, has been implicated in this last step in E. coli (82). However, the

RNA polymerase can in some instances terminate accurately without rho.


Prokaryotic polymerases and regulation of transcription

Two basic mechanisms, negative control and positive control, appear

to be equally effective in controlling prokaryotic transcription. In

negative control, the regulator gene product (a repressor molecule) in

its active form binds to the operator gene to prevent the transcription

of the operon's structural genes. When the repressor is inactivated by

the specific inducer, transcription of the structural genes may proceed.

Thus the repressor controls RNA synthesis by negatively affecting RNA

polymerase activity with respect to specific sites on DNA. The classic

example of negative control is the lac operon of E. coli (83). The lactose

operon is under the negative control of the lactose repressor which binds

specifically to the lactose operator (84,85) region of the DNA, thus pre-

venting transcription of the DNA past the operator. The expression of

this operon requires induction by a galactoside. The inducers bind to

the repressor and decrease the repressor's affinity for the lactose operator.










The histidine operon is another example of regulation by negative

repression, with histidyl-tRNA as the co-repressor necessary for activa-

tion of the histidine repressor which binds to the histidine operator

region of the DNA and prevents transcription of the DNA past the operator (86).

In positive control the RNA polymerase has low affinity for the

promoter site, and the controlling molecule in its active form helps the

RNA polymerase to initiate transcription by increasing the enzyme's

affinity for the promoter site. Positive control of gene expression has

been demonstrated for the arabinose operon of E. coli (86). The protein

product of the C gene is a repressor in the absence of the specific in-

ducer, arabinose. However, in the presence of arabinose the C gene

protein is converted to an activator which is required for expression of

the arabinose genes.

The sigma subunit of RNA polymerase and the catabolite gene activator

protein (CAP) are examples of protein factors which influence transcription

by increasing the affinity of the RNA polymerase for promoters. Sigma

interacts with the core RNA polymerase and facilitates the binding of the

enzyme to a promoter. CAP, also known as cyclic AMP receptor protein,

plays a role in the regulation of genes subject to catabolite repression.

CAP appears to work in addition to sigma to alter initiation specificity

in the presence of cyclic AMP. Cyclic AMP combines with CAP and produces

an allosteric change in the protein. The cyclic AMP-CAP complex then

binds to the DNA close to the promoter of an inducible operon (i.e.,

lactose, galactose, tryptophan, and histidine) and produces a change in

the DNA so that the RNA polymerase can bind to the specific promoter

region and carry out a round of transcription of the operon in question.

Thus the expression of the lactose operon requires cyclic AMP, CAP, holo-

enzyme and the removal of the repressor from the operator.










Alteration of RNA polymerase by phage infection and sporulation

The structure and selectivity of the bacterial RNA polymerase is

altered under certain conditions of viral infection and sporulation. The

alterations of the selectivity of RNA synthesis occur during lytic growth

of the phage in different stages, giving rise to different transcripts

during the course of phage replication and assembly. The bacteriophage T4

infects E. coli and subsequently host RNA synthesis is inhibited, phage-

specific macromolecular synthesis is initiated and the host cells are

ultimately lysed (87,88). A temporal sequence on phage-specific mRNAs

have been detected (89). The first class of mRNA (immediate early)

appeared immediately after infection, and hybridized exclusively to one

strand of T4 DNA. The second class of mRNA (delayed early) to appear

hybridized to later sequential sites on the same T4 DNA strand. The late

mRNA apparently coded for viral coat protein and it hybridized to the

opposite T4 DNA strand.

The sequence of transcription is mediated by changes in the RNA

polymerase. Sigma factor disappears soon after infection (90) and it is

this loss which is presumably responsible for restriction of the tran-

scription of the host genome. There is then a sequential alteration of

each of the subunits of the core RNA polymerase (26,91,92). The subunit

is modified by the covalent attachment of AMP to the alpha subunit. The

RNA polymerase acquires four small T -specific proteins (93) of 10,000-

25,000 daltons which are coded for by the delayed early genes. Thus the

transcription specificity appears to be induced by modifications in the

polymerase or associated factors.

Bacteriophage lamda can either lysogenize or grow lytically (94,95).

During the lytic growth the host E. coli RNA polymerase transcribes the










phage N and Q genes. The two protein products of these genes then mediate

the further transcription of the lamda DNA (96). It appears that the N

gene product is an antiterminator protein that permits the continued

transcription past a rho dependent termination sequence (97). The Q

gene product is a repressor that binds to the operator region for the

lamda repressor gene, thus preventing the transcription of this gene.

Bacillus subtilis phages SPOL and SP82 show a regulated program of

transcriptional alteration during normal growth (98,99). The infected

cell RNA polymerase contains two proteins not present in the normal RNA

polymerase and the sigma subunit is not required for selectivity (100).

Several observations suggest there are specific changes in the

structure of Bacillus subtilis RNA polymerase during sporulation. The

purified sporulating RNA polymerase lacks sigma factor and contains a

modified P subunit of lower molecular weight than the vegetative enzyme

(19,101). In addition, a new polymerase subunit of 60,000-70,000 daltons

appears (102). These modifications appear to be due to the action of

proteases that are present in cells undergoing sporulation, and it is

thought that these changes are related to the alterations in transcrip-

tive specificity that occur during sporulation.

Genetic studies also support a specific role of polymerase in the

sporulation process. Mutants which contain an altered beta subunit fail

to sporulate (101). The evidence form the genetic studies and the bio-

chemical studies is consistent with the hypothesis that changes in the

subunit structure of RNA polymerase mediate the changes in transcription

associated with Bacillus subtilis sporulation.











Eukaryotic RNA Polymerases


Background


There are several recent review articles on eukaryotic RNA polymerases

(47-52). These may be consulted for discussions that are omitted in this

review. The DNA dependent RNA polymerase activity was first demonstrated

in rat liver nuclei by Weiss in 1955 (103) who partially purified the en-

zyme which was firmly attached to DNA. Mans and Novelli (104) were the

first to study a solubilized eukaryotic RNA polymerase in 1964. However

the problem of freeing the RNA polymerase from DNA was more severe for the

other eukaryotic RNA polymerases studied and consequently most studies on

nuclear RNA polymerase activity were carried out on isolated nuclei or

unpurified chromatin until a eukaryotic RNA polymerase was purified in

1965 (105).

Isolated nuclei demonstrated transcription activity which synthesized

mostly GC-rich ribosomal RNA at low ionic strength. This activity was

localized in the nuceolus and stimulated by Mg However, at high ionic

strength mostly DNA-like RNA was synthesized by an activity which was
-H-
stimulated by Mn+ and localized in the nucleoplasm (106-111). The nucleo-

plasmic activity was specifically inhibited by a-amanitin (3).

Multiple forms of nuclear RNA polymerases were demonstrated by Roeder

and Rutter (5) in rat liver and Xenopus laevis. They demonstrated three

types of eukaryotic RNA polymerases which could be separated based on

chromatographic properties, sensitivity to specific inhibitors and sensi-

tivity to ions. The first.RNA polymerase eluted from a DEAE Sephadex

column (nuclear RNA polymerase I) was found to be of nucleolar orgin,

insensitive to a-amanitin and synthesized a product which hybridized to










RNA competitively with ribosomal RNA (rRNA) but not with heterogeneous RNA

(Hn RNA). RNA polymerase II, the second enzyme eluted from the column,

was a nucleoplasmic, a-amanitin sensitive RNA polymerase that synthesized

a product that hybridized to DNA competitively with Hn RNA but not with

r-RNA (32). RNA polymerase III was thought to be nucleoplasmic. Since

then several laboratories have demonstrated that the nucleus, nucleolus

and cellular organelles have unique transcriptional systems (44).

On a protein percentage basis, the amount of RNA polymerase activity

in eukaryotic cells is much lower than in prokaryotes. The RNA polymerases

from calf thymus, which is a tissue rich in the enzyme (112), is only ob-

tained in a few milligrams per kilogram of tissue (113-115). This yield,

which is a few orders of magnitude lower than that to Escherichia coli RNA

polymerase (67,70,116) demonstrates one of the difficulties encountered in

studying the regulation of eucaryotic transcription at the molecular level

(studies which require significant quantities of the highly purified enzyme).

The specific activity of the eukaryotic RNA polymerases is in the order of

that of the purified E. coli RNA polymerase (50,113,114,116).


Mitochondrial transcription

The mitochondrion represents a semi-autonomous organelle within the

eukaryotic cell in the sense that it contains a unique DNA that is geneti-

cally active; it is replicated and transcribed within the organelle to

provide products unique from those found elsewhere in the cell. The mito-

chondrial DNA has been well characterized physically and chemically (117-

122) and the size of the mitochondrial genome varies from a molecular

weight of 10 daltons in vertebrates to 11.9 x 10 daltons in plants

(119,122). DNA with these molecular weights can code for only 5,000 amino












acids or about 20 proteins. Therefore, it is impossible for the mitochon-

drial genome to code for all of the unique mitochondrial proteins which

must carry out replicative, transcriptive, translative and metabolic func-

tions unique to the mitochondrion. All available evidence suggests that

the mitochondrial DNA carries information for the mitochondrial ribosomal

RNA, for at least some mitochondrial tRNAs and for a few mitochondrial

membrane proteins (121,123). Thus mitochondrial DNA is required for the

biogenesis of mitochondria.

The majority of proteins unique to the mitochondrion are coded for

by the nuclear DNA and synthesized on the cytoplasmic ribosomes. Less

than 10% of the mitochondrial proteins are synthesized on mitochondrial

ribosomes; this fraction consists of probably not more than eight species

of hydrophobic proteins that are incorporated into the inner membrane

where they assemble cytochromes, cytochrome oxidase and ATPase (120,

121, 123-126). A few components of the mitochondrial protein synthesizing

system may be coded for by the mitochondrial DNA, although there is no

evidence for the location of the code for the mitochondrial leucyl-tRNA

synthetase (127) and the two mitochondrial peptide chain elongation

factors, G and T (128). However, the prokaryotic characteristics of the

mitochondrial protein synthesizing system makes it difficult to imagine the

code coming from elsewhere in the cell.

The only mitochondrial gene products that have been positively identi-

fied are ribosomal RNA and transfer RNA (121,123). Hybridization experi-

ments have shown that mitochondrial ribosomal RNA hybridizes specifically

with mitochondrial DNA (129-132) and shares no sequence homology between

some of the mitochondrial 4s transfer RNA and mitochondrial DNA (133,134).

In order to understand the extent to which a mitochondrion depends upon










the rest of the cell, a more complete understanding of the mechanisms

which provide unique informational molecules must be examined in detail.

Mitochondrial DNA transcription has been studied in vivo using intact

growing cells and in vitro with isolated mitochondria or solubilized and

partially purified mitochondrial RNA polymerase. These investigations

have provided results which will be useful for the understanding of the

regulatory mechanisms which couple the genetic activities of mitochondria

and nuclei during cell growth and development.

The synthesis of mitochondrial RNA in intact cells has been exten-

sively studied in HeLa cells by Attardi and coworkers. Hybridization

experiments of in vivo labeled mitochondrial RNA with the separated H

and L strands of mitochondrial DNA demonstrated that both the H and L

strands are equally transcribed but afterwards the product of the L

strand is almost completely degraded. This means that the problem of

strand selection in mitochondrial transcription could be achieved by

transcribing both strands and rapidly degrading or exporting 98% of the

L-strand transcripts (135-140). The transcription products have been

used to map the mitochondrial DNA; the H-strand contains genes for 9

tRNAs, whereas the L-strand has the two ribosomal RNA genes and also

those for three tRNAs (137-141). Election microscopy of ferritin-

labeled tRNA-mitochondrial DNA hybrids confirmed the location of these

genes (142). Experiments with the HeLa cells also suggest that mito-

chondrial messenger RNA is also translated. Mitochondrial RNA containing

poly (A) hybridizes with mitochondrial DNA (142).

Studies of in vitro incorporation of ribonucleotide triphosphates

into high molecular weight RNA have shown that isolated mitochondria











are capable, for some time, of carrying out RNA synthesis just as are

mitochondria within the living cell, except that the process appears to

be much slower in vitro than in vivo. The incorporation of UTP or ATP

generally requires all four ribonucleotide triphosphates and magnesium

(143,144). The incorporation process is considerably faster if the mito-

chondria are swollen. Mitochondrial RNA synthesis is inhibited by

actinomycin D, which binds to the DNA template, proving its dependence

on DNA. The product of the in vitro incorporation reaction hybridizes

with the H strand of mitochondrial DNA (145). In Xenopus mitochondria

the RNA polymerase initiates at several different sites of the template

predominantly with ATP and 2-fold less frequently with GTP (146).

Studies on mitochondrial RNA synthesis have recently concentrated

on the characterization of the DNA-dependent RNA polymerases. However,

relatively little is known about the properties and functions of the

mitochondrial enzymes as compared to the nuclear RNA polymerases. It

has been difficult to identify and isolate the mitochondrial RNA poly-

merase due in part to the difficulty of defining proper conditions for

the solubilization of this enzyme which appears to be firmly bound to

mitochondrial membrane. Studies on mitochondrial RNA polymerases from

a variety of sources have shown that these enzymes exhibit properties

clearly distinguishing them from the respective nuclear RNA polymerases.

The mitochondrial enzyme differs from the nuclear RNA polymerase II

in sensitivity to a-amanitin (11, 147, 148, 149). Low concentrations of

a-amanitin (0.1 to 20 pg/ml) which cause inhibition of nuclear RNA

polymerase II does not affect the mitochondrial RNA polymerase. Although

some functional differences have been described in mitochondrial RNA

polymerases, there is general agreement regarding their size.










The molecular weight of the enzyme, consisting of a single polypep-

tide chain, seems to be about 60,000 (11-15). The purified mitochondrial

RNA polymerases from Neurospora crassa (11) and rat liver (12,13,147) are

inhibited by rifampicin in contrast to the mitochondrial enzymes from

Xenopus laevis (14), wheat leaf (150) and Ehrlic Ascites (39) which are

resistant to rifampicin. Some investigators have found the yeast mito-

chondrial RNA polymerase to be rifampicin resistant (15,148,151-153).

Others describe the isolation of rifampicin-sensitive enzyme (154-155).

The reason for this discrepancy is not clear. It is possible that a

factor or factors needed for conferring sensitivity to rifampicin were

lost from some enzyme preparations.

In contrast to the above studies several laboratories have reported

that the yeast mitochrondrial RNA polymerases are much larger in size and

resemble more closely the nuclear polymerases. Eccleshall and Criddle

demonstrated three RNA polymerases associated with yeast mitochondria

having molecular weights near 500,000 and showing no sensitivity to

rifamycin (153). However, there was no difference between the nuclear

RNA polymerase I and the mitochondrial RNA polymerase I, suggesting that

their mitochondrial preparation was contaminated with nuclear enzyme.

Reports of multiple RNA polymerases associated with yeast mitochondria

having molecular weights near 500,000 and showing no sensitivity to

rifamycin have come from Rogall and Wintersberger (15) and Benson (156).

Scragg has reported a yeast mitochondrial- RNA polymerase with molegular

weight greater than 200,000 that does show a rifamycin sensitive enzyme in

yeast (157). However, despite the low molecular weight (67,000) of the

yeast mitochondrial RNA polymerase polypeptide isolated by Rogall and

Wintersberger (15), this enzyme readily forms aggregates with molecular










weights up to about 500,000 in buffers of low ionic strength. The same

is true for the 64,000 molecular weight enzyme purified by Kuntzel and

Schafer (11) and the 60,000 molecular weight enzyme isolated by Scragg

(158). The overall picture of mitochondrial RNA polymerase is one of

either a great diversity of enzyme types associated with different organ-

isms or else some differences in the isolation procedures employed in the

various laboratories which give rise to preparations with these differences.

The mitochondrial RNA polymerase consisting of a single polypeptide chain

of molecular weight of 64,000 resembles more the bacteriophate T7 specific

RNA polymerase rather than E. coli or eukaryotic nuclear RNA polymerase.

The T7 enzyme is a product of T7 DNA gene 1. A single polypeptide of

110,000 molecular weight, it generates only eight RNA species in late

transcription of T7 DNA (25).


Chloroplast transcription

The chloroplast also contains unique DNA that is replicated and

transcribed within the organelle. The molecular weight of Euglena

chloroplast DNA is approximately 108 daltons (159) which is sufficient

to code for the chloroplast tRNA, rRNA, and mRNA. However, the extent

to which the total potential genetic content is expressed in chloroplast

is open to question. It has been established that the chloroplast DNA

contains cistrons coding for chloroplast ribosomal RNA (160,161) and

transfer RNA (179). Quantitative saturation-hybridization of Euglena

chloroplast rRNA to chloroplast DNA showed that approximately 2% of the

DNA is involved in coding for chloroplast rRNA (160). A higher value (6%)

was obtained by Stutz and Vandrey (161) who isolated Euglena chloroplast

DNA from purified organelles devoid of any nuclear contamination rather










than relying on the fractionation of extracted DNA by preparative CsC1

buoyant density centrifugation. The chloroplast ribosomal 23S RNA (1.1 x

106 daltons) and 16S RNA (0.56 x 106 daltons) are coded for by separate

cistrons (162) localized on the heavy strand of the chloroplast DNA.

Accepting that the molecular weight of Euglena DNA is 108 daltons then

2% of this (2 x 106 daltons) is sufficient to code for both the 23S and

16S rRNAs. The value of 6% would allow for three copies of each of the

rRNAs per chloroplast DNA molecule.

The presence of mRNA that can be translated has been demonstrated

in Euglena chloroplast (163). These mRNAs could be the products of

transcription of chloroplast DNA or of nuclear DNA, or of both. However

isolated chloroplasts synthesize RNA and such RNA must necessarily be

chloroplast-DNA coded. Hybridization of RNA synthesized in isolated

tabacco chloroplasts to chloroplast DNA indicated that 21% of the

chloroplast DNA is transcribed (164). This represents a much larger

proportion of the chloroplast DNA than is required to code only for

ribosomal and transfer RNAs and therefore implies that mRNA species are

synthesized on chloroplast DNA.

Chloroplasts of higher plants and algae were shown to contain a

DNA-dependent RNA polymerase as early as 1964 but the enzyme was only

recently solubilized and purified (165). The extreme difficulty with

solubilizing this activity was related to the tight binding of the enzyme-

DNA complex to the thylakoid membranes. This chloroplast enzyme contained

two large polypeptide subunits, which suggested a similarity to the

prokaryotic and the eukaryotic nuclear RNA polymerase rather than the

mitochondrial or bacteriophage T7 RNA polymerases. The chloroplast enzyme

has also been studied in Euglena gracilis (166). The Euglena chloroplast










RNA polymerase has not been solubilized and purified mainly due to the

difficulties mentioned above. This prompted the study of chloroplast

transcription complexes consisting of chloroplast membrane fragments,

DNA, and the RNA polymerase activity.


Euglena RNA polymerases

The nuclear RNA polymerase II of Euglene gracilis was partially

purified recently by Congdon and Congdon and Preston (167,168). This

enzyme, defined by its sensitivity to a-amantin, eluted from DEAE

Sepahdex between 0.18 and 0.21 M (NH4)2SO4. The RNA polymerase had a

broad salt optimum ranging from 50 mM (NH )2SO4 to 150 mM (NH )2SO0.

Optimal activity was exhibited with optimum MnCl2 concentrations rather

than with MgCl2. The enzyme was more active with poly d(AT) as a

template and preferred denatured over native calf thymus DNA. The

Euglena RNA polymerase, as well as the enzymes from other protests,

were less sensitive to a-amantin than the corresponding fractions from

higher eukaryotes. The Euglena chloroplast and mitochrondrial RNA poly-

merases have not been previously solubilized and purified.

















MATERIALS AND METHODS


Buffers

All buffers were prepared from analytical reagent grade chemicals

without further purification. Double distilled or deionized water was

used for all solutions. Stock solutions of 1.0 M Tris-HC1 pH 7.4, at

250C, 1.0 M Tris-HCl pH 7.9, at 250C, 1.0 M Tris-HCl pH 9.0, at 250C,

0.10 M EDTA pH 7.6, 1.0 M MgCl2, 0.01 M MnC1, 0.10 M dithiothreitol were

diluted to prepare the following buffers:

Buffer STE; 0.25 M sucrose, 0.01 M Tris-HC1 pH 7.4 or pH 9.0,
0.10 mM EDTA pH 7.6.

Buffer STM; 0.25 M sucrose, 0.01 M Tris-HCl pH 7.9, 0.01 M
MgC12.

Lysis buffer; 0.01 M Tris-HCl pH 7.9,20% w/v sucrose. 0.10
M KC1, 5 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride.

Buffer TGMED; 0.05 M Tris-HC1 pH 7.9, 25% glycerol, 5.0 mM
MgC12, 0.10 mM EDTA pH 7.6, 2.0 mM dithiothreitol.

Ethidium bromide buffer; 1 Vg/ml Ethidium bromide, 0.01 M
Tris-HCl pH 7.9, 0.02 M NaCI, and 0.005 M EDTA.


Organism

Premanently bleached mutants of Euglena gracilis klebs strain z,

prepared by treatment with streptomycin (169) were used for these studies.


Growth

The streptomycin bleached mutants were cultured in 2800 ml Fernback

flasks containing 1500 ml of Medium A described by Greenblatt and Schiff (170).










The cultures were grown at 270 on a gyrotary shaker operating at 120

rev/min. Under these conditions, cells enter stationary phase when the

carbon source is exhausted (171). Growth was measured as turbidity with

a Klett colorimeter with a No. 66 filter.


Cell disruption and subcellular fractionation

Cells from axenic cultures in exponential phase were harvested by

passing the contents of the culture flaks through a DeLaval cream separator

centrifuge operating at room temperature. All further operations were

performed at 00 -40 C.

The cells were washed twice in STE buffer, pH 7.4, then suspended

in at least 5 volumes of STE buffer, pH 9 and passed through an Aminco

French Pressure cell at 1000 lb/inch2 (100) using a power press to give

greater than 90% breakage. Breakage was estimated by microscopic examina-

tion. The cell lysate was collected in ice chilled flaks, shakengently

to disperse aggregates, and repeatedly centrifuged at 1000 x g for 5 min

(Beckman J-21 JA-20 rotor) until only a negligible pellet was produced.

Crude subcellular fractions were collected from the cell free lysate

supernatant by differential centrifugation first at 5000 x g, 20 min and

then 10,000 x g for 15 min. The crude 5000 x g pellet (5P mitochondria)

was washed in STE pH 7.4 buffer.


Mitochondrial purification

Washed 5P mitochondria were purified in gradients of sodium diatri-

zoate (Winthrop Laboratories, New York). Samples containing 5-200 mg

dry weight of mitochondria suspended in the less dense renografin solution

(density equals 1.103 g/cm3) containing 0.01 M Tris-Hcl pH 7.4 and 0.10 mM










EDTA were layered over the Renografin gradients (density equals 1.103 to

1.30 g/cm3) containing 0.01 M Tris-HCl pH 7.4 and 0.01 mM EDTA. The pre-

parations were centrifuged at 20,000 rpm for 2 hr at 20C (Beckman L-2,

SW 25.1 rotor). Fractions were collected and the mitochondria banding

in the region of the gradient corresponding to a density of 1.21 g/cm3

were washed twice in STE buffer pH 7.4 before use in incorporation studies.

Washed 5P mitochondria were alternatively purified by the enzymatic

digestion of nuclear DNA with 50 pg deoxyribonuclease I (E. C. 3.1.4.5) per

ml. The crude 5P mitochondria were washed in STM buffer pH 7.9 and then

pancreatic DNase I (Sigma Chemical Co., St. Louis, Mo.) was added to a

final concentration of 50 pg per ml in STM buffer. Incubation was carried

out at 40 for 30 min. with slow stirring. The enzymatic digestion was

stopped by the addition of 2 volumes of 10% sucrose, 0.01 M Tris-HC1 pH

7.9,0.05 M EDTA pH 7.6. The mitochondria were then washed in STE buffer

pH 7.9.


Succinic dehydrogenase assay

Succinic dehydrogenase activity was measured by the succinate depend-

ent reduction of dichlorophenolindophenol (172) in 1 ml reaction mixtures

at room temperature.


Extraction of nucleic acids

DNA was extracted from the mitochondria preparations by the technique

of Bhargava and Halvorson (173).


Characterization of DNA

The method of Preston and Boone (174) was used for the density measure-

ment of DNA. The CsC1 gradient polyacrylamide gels obtained after the










exposure to light were washed with flowing tap distilled water for 2 hr

to remove CsCl. The riboflavin was photooxidized by shaking the gels

under illumination for 16 hr in the presence of 10 ml of 0.01 N NaOH in

50% formamide. The gels were then washed in tap distilled water for 2

hr before adding the gels to ethidium bromide staining buffer. The

staining permitted the quantitative detection of DNA in the gel which

could be scanned in an Aminco Flourometer equipped with a gel scanning

attachment.


DNA standards

DNA was purified from Cytophage P-ll (a strain isolated, identified

and carried at the Department of Microbiology, University of Florida,

Gainesville, FL) as described by Preston and Boone (174). DNA from the

bacteriophage 025 and DNA from Pseudomonas aeruginosa were kindly pro-

vided by M. Mandel, M. D. Anderson Tumor Institute, Houston, Texas.

DNA was extracted from isolated nuclei of streptomycin bleached mutants

of Euglena gracilis Klebs strain z by the method of Bhargava and Halvorson

(173).


Preparation of cellular fractions for protein and DNA analysis

Cellular fraction samples were prepared for analysis by Vortex mixing

1 ml of sample with 5 ml of cold 95% ethanol (-200C). The particulate

material was collected by centrifugation at 4000 rpm, 5 min., 20C. The

ethanol extraction was repeated twice more before suspending the pellet in

1 ml of cold double distilled water. The samples were suspended in 1.0 ml

of water then 4.0 ml of cold 0.50 N HC104 was added and the sample was

mixed on a vortex. The precipitated macromolecules were collected by

centrifugation before being suspended in 4 ml of cold ether-ethanol (1:3)










and centrifuged again. The pellet was suspended in 4 ml of 0.50 M HC104

and incubated at 850C for 20 min. to hydrolyze DNA and RNA, cooled and

centrifuged. The top 3 ml of the supernatant was used for DNA analysis.

The pellet was suspended in 3 ml of 1.0 N NaOH and heated in a boiling

water bath until the precipitated protein was dissolved. This solution

was used for analysis of soluble proteins.


Protein determination

The protein concentrations were determined by the Lowry method (175).

Dissolved bovine serum albumin served as the standard.


DNA determination

DNA concentrations were determined by the indole method of Hubbard

(176). Calf thymus DNA served as a standard. A sample of 0.05 ml was

added to 0.05 ml of 10% trichloroacetic acid and 1 ml of 0.04% indole-2

N HC1 solution. The mixture was shaken and incubated at 970C for 15 min.

and then cooled in ice. The mixture was then extracted with chloroform

three times by adding 2 ml of CHC13 to the mixture, vortexing and centri-

fuging 2000 rpm, 5 min. The chloroform phase was removed after each ex-

traction. The absorbance of the aqueous phase at 490 mm was then determined.


RNA polymerase assay

Conditions for the assay of RNA polymerase were similar to those

described by Preston et al. (177). The routine mixture contained in a

total volume of 0.10 ml: 0.05 M Tris-HCl pH 7.9, 0.005 M MgC12, 0.001

M MnC12, 0.001 M DTT, 0.01 M KC1, 0.001 M of each unlabeled ribonucleoside

triphosphate, 0.032 mM or 0.004 mM or 0.016 mM (2 Ci/mmole of 3H-labeled

ribonucleoside triphosphate, 100 pg/ml of denatured calf thymus DNA and

enzyme. Assay mixtures were incubated for 10 min. (or the time indicated)











at 370C. The reaction was stopped by adding stop bath (0.05 M sodium

pyrophosphate, 2 mg/ml bovine serum albumin, 2 mg/ml Torula RNA and 5 mM

of the unlabeled ribonucleotide which was identical to the labeled NTP)

and chilling in ice (1). Macromolecules were precipitated by adding 2

ml of cold 10% trichloroacetic acid containing 20 mM sodium pyrophosphate

(TCAPP) to each reaction tube. After standing in ice for 15 min. the

precipitate was collected by filtration through a glass fiber disc (What-

man GF/C) which had been prewashed with 5 ml of cold TCAPP. Each disc

was washed three times with 5-10 ml TCAPP, then three times with 5-10 ml

ether-ethanol (1:3) 370C, followed by two washes 5-10 ml ether.

Filtration was carried out in perforated porcelain crucibles (Gooch)

adapted to a vaccum manifold with ruber crucible holders (Walter).

Filtration was facilitated by maintaining a partial vaccum (ca. 28 in.

Hg). The GF/C filters were dried and placed in scintillation vials

containing 5 ml of toluene (scintillation grade, Mallinkrodt), 0.4% PPO

(2,5-diphenyloxazole), 0.01% POPOP 1,4-di[2-(5-phenyloxazolyl)]-benzene),

and counted for 10 min. in a liquid scintillation counter (Beckman LS-133).

A unit of enzyme activity is defined as that which will catalyze the

incorporation of 1 pmole of labeled ribonucleoside monophosphate into acid

precipitable material in 10 min. at 370.


Isolation of 3H CTP labeled product

A sample of 0.30 ml of purified mitochondria (0.18 mg/ml protein)

was incubated in a 3.0 ml RNA polymerase reaction mixture (0.05 mM 3H-CTP,

2 Ci/mmole) for 15 min. The reaction was stopped by adding 3.0 ml of stop

bath (0.05 M sodium pyrophosphate, 1.09 mg/ml E. coli RNA and 5 mM of

unlabeled CTP) and brought to 0.20 M NaC1 and 0.10 M Tris pH9. Triton X











100 was added to bring the mixture to a final concentration of 0.5%. The

mixture was then mixed with an equal volume of 80% v/v redistilled aequeous

phenol. The nucleic acids were collected by precipitation with 3 volumes

of cold 95% ethanol (-200C) for 12 hr followed by centrifugation at 4000

rpm 15 min. The nucleic acids were dissolved in buffer (0.20 M NaC1, 0.10

M Tris pH 9) and carried through the ethanol precipitation again. The

final pellet was dissolved in 2 ml of double distilled water.


Column filtration

Biogel P-4 was swollen overnight by mixing 1 g Biogel per 10 ml of

0.10 M NaC1. A 20 ml column was poured and pre-equilibrated by passing

4 volumes of 0.10 M NaC1 through it at 40C. Nucleic acid samples in

0.10 M NaCl were added to the column and eluted with 0.10 M NaC1. The

flow rate was 10 drops per min. 3H labeled samples (100 pl) were placed

in 10 ml of Brays scintillation fluid (178) and counted in the liquid

scintillation counter (Beckman LS-133).


Extraction of phospholipids

Phospholipid extraction was performed by the method of Carter and

Kennedy (179) and Raetz and Kennedy (180). To 0.10 ml of the 3H-CTP

labeled product was mixed 1.0 ml of methanol containing 0.10 N HC1. Ten

minutes later 2 ml of chloroform was added and the phases were stirred

with a vortex mixer before adding 4 ml of 2 M KC1. The mixture was then

vigorously mixed for 5 min. and the phases were allowed to separate before

the aqueous phase was removed. The chloroform phase was washed two more

times with 2 M KC1. A sample of each phase following extraction was

placed in a scintillation vial and dried by passing a stream of nitrogen










gas over the solution. Then 10 ml of Bray's solution was added to the

vials and counted.


Thin layer chromatography methods

The procedure of Ter Schegget et al. (181) for thin layer chromato-

graphy of CDP-diglyceride was followed. Samples were spottedon silica

gel G thin layer plates. Chromatograms were developed with a solvent of

chloroform-methanol-water-ammonia (70:38:2:8). The CDP-diglyceride stand-

ard was prepared by the method of Carter and Kennedy (179). Nucleotides

were chromatographed on PEI cellulose by the methods of Randerath and

Randerath (182,183). Ribonucleoside monophosphates were chromatographed

first in 1.0 N acetic acid to a height of 4 cm and then without drying

the plate was transferred to a tank containing 0.30 N LiC1 and the solvent

was allowed to rise to 15 cm. The ribonucleoside triphosphates were

separated by developing to 15 cm with 0.50 M ammonium sulfate.


Solubilization and purification of
mitochondrial RNA polymerase

Solubilization of mitochondrial RNA polymerase was achieved by fol-

lowing a procedure similar to that of Wintersberger and Rogall (15, 152).

Mitochondria from 150 g wet weight of cells were suspended in an equal

volume of lysis buffer. This was added to an equal volume of a lysis

mixture consisting of 1.6% Triton X 100 non ionic detergent, 1.0 M KC1,

and 0.01 M EDTA (pH 7.9). The mixture was mixed gently for 15 min and

then centrifuged for 90 min at 30,000 rmp (Ti 50 rotor) and 0.29 g of

solid ammonium sulfate per ml of 30,000 supernatant was added to bring

the extract to 50% saturation with respect to ammonium sulfate. The

precipitate was collected by centrifugation at 40,000 rmp for 90 min.











(Ti 50 rotor) and dissolved in TGMED containing 0.05 M (NH)2SO4 and

dialyzed against 2 liters of the same buffer for 4 hr. The dialyzate

was applied to a column (0.90 x 20 cm) of DEAE-Sephadex A-25 which had

previously been equilibrated with the same buffer and nonabsorbed protein

was washed off the column with 30 ml of the same buffer. The RNA polymerase

activity was eluted with a 160 ml gradient of 0.05 M to 0.50 M (NH )2SO4

in TG,ED. Fractions (2.50 ml) were collected and 20 pl of each fraction

was assayed for RNA polymerase activity. Concentrations of (NH4)2SO4 were

determined with a conductivity bridge.


Chemicals and reagents

The following research grade biochemicals were obtained from Sigma

Chemical Co., St. Louis, Mdo: The ribonucleoside triphosphates GTP, CTP

UTP and ATP as sodium salts; dithiothreitol; phenylmethylsulfonyl flouride;

high molecular weight calf thymus DNA; Torula yeast RNA, Triton X-100; en-

zyme grade trizma buffers; chromatographically pure pancreatic ribonuclease

A (EC 2.7.7.16) and deoxyribonuclease I (EC 3.1.4.5): fraction V bovine

serum albumin; cytidylyl (3'+ 5') cytidine (CpC); and snake venom phospho-

diesterase (Crotalus atrox venom, Type VII).

The tridium labeled ribonucleoside triphosphates as sodium salts

were purchased from Schwartz/Mann, Orangeburg, NY. These include {5-3H}

UTP, {5-3H} CTP, {8- H} ATP, {8- H} GTP. Ethidium bromide and actinomycin

D were purchased from Calibiochem, LaJolla, CA. Poly d(AT) was obtained

from Miles Laboratories, Kankakee, IL. The rifamycin derivatives AF/05

and AF/013 were the generous gift of Gruppo Lepetit, Milan, Italy. The

a-amanitin was kindly provided by Dr. T. Wieldand, Max-Planck Institute,







33


Heidelberg, West Germany. Sodium diatrizoate was purchased from Winthrop

Laboratories, NY. Nonionic detergent NP-40 was purchased from Shell Oil

Co. Unless specifically noted, all other chemicals were analytical rea-

gent grade.

















RESULTS


Two experimental approaches were utilized to study the in vitro mito-

chondrial RNA synthesis in Euglena gracilis. The first approach was to

study the incorporation of radioactively labeled ribonucleoside triphos-

phates into acid insoluble products by isolated mitochondria which had

been treated with isotonic buffer to assure swelling and permability.

The products of the incorporations were then studied. The second approach

was to study the activity of solubilized and partially purified mitochon-

drial DNA dependent RNA polymerase. This approach involved determining

the conditions for solubilizing the enzyme and then for partially purifying

the enzyme. The activity of this enzyme was then characterized.


The Incorporation of Ribonucleoside
Triphosphates by Isolated Mitochondria

The initial research objectives were to determine if RNA synthesizing

activity is retained by mitochondria isolated from Euglena gracilis, and

to look for ways to differentiate between nuclear and mitochondrial RNA

polymerase activities such as sensitivities to inhibitors or special

enzymatic requirements. The approach was to study the RNA synthesizing

activity of isolated mitochondria obtained from a streptomycin bleached

aplastidic mutant of Euglena gracilis, strain z. The mitochondrial

isolation procedure (Figure 1) consisted of rupturing washed cells in the

French pressure cell at 1000 lb/in2 and collecting crude mitochondrial




























Figure 1. SCHEMA FOR THE ISOLATION OF MITOCHONDRIA













ISOLATION


WASHED


PROCEDURE


CELLS
STE (0.25M Sucrose, O.O0M Tris-HCI pH7.6,
O.ImM EDTA pH 7.4)
--Fpc 1000 Ib/inch


DISRUPTED CELLS


--1000 Xg, 5 min.
REPEAT I-3X


5000 Xg, 20 min.


resusp.
5000X g, 15


Washed 5P
(Dense Mito.)

RENC


5S


IC


10000 >
min.

)P 10S


(g, 15 min.


(Less Dense)
(Mito.)
)GRAFIN GRADIENT


MITO. (p= 1.22 g cm-3)


PURIFIED










pellets by differential centrifugation, first at 5000 x g and then at 10,000

x g for 15 min. The crude 5000 x a mitochondrial pellet (5P) containing

the heavier and more enzymatically active mitochondria (33) were used for

these experiments. The 5P mitochondria were washed in buffer and then

subjected to sodium diatrizoate (renografin) gradient centrifugation at

20,000 x g for 2 hr. Fractions were collected and the mitochondria, which

banded in the region of the gradient corresponding to a mean equilibrium

density of 1.22 g/cm3 were used for the studies reported here.

In order to test the purity of the mitochondrial preparation, the

buoyant density of the DNA in the mitochondrial preparations was routinely

monitored by CsC1 gradient centrifugation. The CsC1 gradients were genera-

ted in the presence of acrylamide and catalyst according to the procedure

of Preston and Boone (174). This method allows the DNA that is banded in

the CsC1 gradient to be fixed by exposing the gradient to light in order to

polymerize the acrylamide gels. Ethidium bromide staining permitted a rapid

and sensitive technique to quantitatively detect the DNA in the gels which

could be scanned in an Aminco Fluorometer equipped with a gel scanning

attachment. The linear relationship between the relative position in the

gradient and the buoyant density of the DNA is demonstrated by the plot of

DNA standards of known densities in Figure 2. The purified mitochondrial

preparation yields better than 70% mitochondrial DNA which has a density

of 1.691 g/cm3 and less than 30% nuclear DNA which has a density of 1.707

g/cm3. Although it is evident that the preparation contains some contami-

nating nuclear DNA, this method provided the cleanest feasible mitochondria

without treating the mitochondria with an exogenous nuclease.
















Figure 2. DENSITY EQUILIBRIA ANALYSIS OF MITOCHONDRIAL DNA


Density equilibria anlaysis of DNA in mitochondria purified on a sodium
diatrizoate (Renografin) gradient were measured by CsC1 density gradient
centrifugation as described in the methods section. The markers are
Cytophage P-1 DNA with a density of 1.693 g/cm3, Psuedomonas aeruginosa
DNA with a density of 1.727 g/cm and bacteriophage d DNA with a density
of 1.742 g/cm .


















CsCI Density Gradient Gel
DNA STAINED WITH ETHIDIUM BROMIDE
M






a) Cytophaga P- I
b) Pseudomonas aeruginoso DNA
c) 025 DNA

LJ
2 N
U n b
Un to
u \ -1.80 1
o : E
O u


aM N .7






1.60


BOTTOM TOP 3
DISTANCE (cm










The renografin purified mitochondria are active biochemically as demon-

strated by results in Table 1. Succinic dehydrogenase which is tightly

integrated into the inner membrane of mitochondria has been employed as a

marker enzyme in order to follow the purification of mitochondria. Of the

total cellular succinic dehydrogenase activity 42% is recovered in the

purified mitochondria, with a 27-fold purification per mg of protein and a

70-fold purification per pg of DNA. This demonstrates that the purification

procedure yields mitochondria biochemically active for an enzyme not asso-

ciated with the DNA


A Mitochondrial Activity Incorporating Label
From 3H-CTP Into Acid Precipitable Product

The incorporation of label from the four ribonucleoside triphosphates

labeled with tritium by isolated mitochondria was studied by performing

RNA polymerase assays as described in the methods section. The mitochondria

were treated with isotonic buffer to assure swelling and permability. The

relative rate with which label from each ribonucleoside triphosphate was

incorporated into acid insoluble macromolecules was determined by performing

the assays in 0.1 ml reaction mixtures in which only one of the ribonucleo-

tides was labeled. It was observed that isolated mitochondria catalyzed

the incorporation of label from 3H-CTP into acid insoluble material at a

rate greater than 10 times that for the other ribonucleotides (Table 2).

It is interesting that ATP and GTP, the ribonucleotides which normally

serve as the best substrate for RNA initiation in procaryotic systems, are

the poorest substrates for the reactions studied here. Figure 3 demonstra-

tes the relative rates of incorporation of tritium from 3H-CTP and 3H-UTP,

the two most active substrates. In this experiment the concentration of


















o Ifr 0

N-
r(~r-


al- a' LC C
L( 0 0\
H0
i-1


O 0o CN
mo -- --T


U-)



w X
H

z


I I N
O r lC


"O O
Ii 0H ,U



o r.'


-H 0 U)
0 r
"o 0
41 -

OZ 0



P 4J "

0H 03
ww *w
0 ) 0
Q 4J *I-
a -i 3




w -o2





m 401
0 0 H


'0 .0
C-1 J 0

t0 a 0

0 0)
4H iu H





J -4 -H
a) "aw .
Ew 0 -


0 0o
O C
0 0 q

CO 4 CO


900





U ) 0 -


3 S 4 0)
4310 U 0







owo
0' (4 ) 4



0 4- CO
X a 0










04 *03 1 -
o 0) 't






0-s *U i

' 0 w o 0
zH 0 HO


'H 0 )-



o4- M 0 C
C H o- *
U3kD(U>
>M- R *i-l


o
C)



H-
HZ

0
O
E-i

















TABLE 2


NUCLEOTIDE INCORPORATION BY
ISOLATED MITOCHONDRIA


CPM incorporated into acid
insoluble macromolecules.


Labeled
Ribonucleotide


3H-CTP 13860

3H-UTP 952

3H-ATP 276

3H-CTP 322



The RNA polymerase assay is described in the methods
section. Each reaction mixture contained in a total
volume of 0.1 ml: 1.0 mM of each unlabeled NTP and
0.004 mM of each H-labeled ribonucleoside triphosphate
with a specific radioactivity of 2 Ci/mmole.
















Figure 3. RIBONUCLEOSIDE TRIPHOSPHATES INCORPORATION BY
ISOLATED MITOCHONDRIA


The incorporation of ribonucleoside triphosphates by isolated mitochondria
into acid insoluble products. The RNA polymerase assay described in the
methods section was followed except that the concentration of the radio-
actively labeled ribonucleoside triphosphates was 0.016 mM, 2 Ci/mmole.
The unlabeled ribonucleotides concentration was 0.5 mM each.











CTP
CTP + RNose
UTP
UTP+ RNase


O 4-
a
o 20-
L 0
CL





o
o 0-



0 10



Z2
c1-1
roo

0 0
10- (ma)





5






5 10 15 20 25
Time (min.)


30





25


--o-
_+_










the radioactively labeled ribonucleotide was 0.016 mM and that of the

unlabeled ribonucleotides was 0.5 mM each. The other components of the

reaction were as described in the methods. Label from 3H-CTP was incorpora-

ted at a rate greater than 10-fold that for UTP. It should be noted that

the CTP and UTP tritium incorporations are plotted on different scales in

Figure 3. When the incubation is carried out in the presence of RNase A, the

product formed with 3H-CTP incorporation shows no sensitivity whereas the

product formed with 3H-UTP shows some sensitivity to RNase A. This could

be due to the possibility that the CTP product is not RNA or that the product

is completed with DNA or folded and completed such that it is RNase insensi-

tive. These results also suggest that RNA synthesis could be better followed

by studying the incorporation with labeled UTP.

The incorporation of tritium from 3H-CTP by the isolated mitochondria

was clearly not dependent upon the presence of the other ribonucleoside

triphosphates which appear to inhibit the incorporation by about 10 percent

(Table 3). Incorporation of label from 3H-UTP also shows no dependence

upon added ribonucleotides while the cpm for ATP and GTP were too low to

make any judgment as to their dependency.

The high rate of incorporation with 3H-CTP relative to that with the

other ribonucleoside triphosphates prompted the investigation of the incor-

poration with 3H-CTP. This activity demonstrated no dependency upon added

exogenous DNA or ribonucleoside triphosphates (Table 4). In fact the isolated

mitochondria incorporated label from 3H-CTP at a 10 times greater rate in the

absence of added DNA or nucleoside triphosphates than when they were present.

The distribution of the incorporating activity with 3H-CTP was followed

through the isolation procedure and the results are shown in Table 5. It was
















TABLE 3


INCORPORATION OF RIBONUCLEOSIDE
ISOLATED MITOCHONDRIA.


TRIPHOSPHATES BY


Labeled
Ribonucleotide CPM/Rx.Mix. Units/g Wet Wt.

H-CTP
+ NTPs 11,513 2161
NTPs 12,792 2401

3H-UTP
+ NTPs 616 116
NTPs 1,386 260

3H-ATP
+ NTPs 206 39
NTPs 324 61

H-GTP
+ NTPs 84 16
NTPs 290 54


The RNA polymerase assay was performed as described in the
methods. 0.1 ml reaction mixtures contained 1.0 mM of each
unlabeled NTP, 0.004 mM 3H NTP, 2 mC/mmole.
















TABLE 4

THE DEPENDENCY OF THE MITqCHONDRIAL ACTIVITY
INCORPORATING LABEL FROM H-CTP UPON ADDED
RIBONUCLEOTIDES AND DNA


Assay Components


Units/mg Protein


Complete Assay with DNA

-DNA + NTP

-DNA NTP

-DNA + UTP

-DNA + ATP

-DNA + GTP


1,500

1,481

11,056

9,611

10,667

8,611


The RNA polymerase assay was performed as described in the
methods. 0.1 ml reaction mixtures contained 1.0 mM of each
unlabeled NTP and 0.004 mM of 3H-CTP with a specific radio-
activity of 2 Ci/mmole.





























r 00


C4

Cn


It~c O' -

\O tI r-


I co o0 c
C4 -


U

U H
C.M

o 0





J .E-I
Ou
o o



4-1




00



r4

C
o
cu







ro











,-) -
SU














r CU
0
c



0 0

0O
n o



,0

U 0









H 0 0>
CO
1 03



C















0 co
E 4
0) 0


h 4-1
(0
? E











><014-
r- *-
0d 3 >
&. -1 l-
0 4-J


CN



H0










4-I
E0 -
0 0














E-4


0
.,










observed that 13% of the activity was recovered in the purified mitochon-

dria with an 8.7-fold purification per mg of protein and 125-fold per Pg

of DNA. This suggests that if the activity is associated with DNA, then

it is with mitochondrial DNA and not nuclear DNA, 95% of which has been

removed by the purification procedure.

The incorporation of 3H-CTP into acid insoluble product is inhibited

by actinomycin D (Table 6) at concentrations above 50 pg per ml, suggesting

that a DNA dependent reaction may be involved. However, it is interesting

that relatively high concentrations are required to demonstrate this

inhibition. The possibility of a secondary site of action of actinomycin

D or inhibition by other compounds in the actinomycin D can not be ruled

out. However, pyrophosphate at 2 moles per ml inhibited the activity

while inorganic phosphate at 2 moles per ml failed to inhibit the incor-

poration indicating that the incorporation involves a pyrophosphorolysis.

This indicates the incorporation of CMP and rules against reactions which

incorporate CMP from CDP such as that of nucleotide phosphorylase or the

incorporation of CDP from CTP.

Rifampin and streptovaricin, inhibitors of initiation and elongation

of prokaryotic RNA polymerases respectively, failed to inhibit the activi-

ty. Rifamycin AF/013, and inhibitor of initiation with eukaroytic nuclear

RNA polymerases as well as prokaryotic RNA polymerases, also failed to

inhibit the activity. Ethidium bromide, which will bind to DNA, showed

only slight inhibition which is probably not significant.

The acid insoluble product is sensitive to snake venom phosphodies-

terase and alkaline hydrolysis (Table 7) under two different conditions,

suggesting that the product contains phosphodiester bonds. Up to this

point the product had demonstrated properties that were similar to those
















TABLE 6

INHIBITION OF 3H-CMP INCORPORATION


Percent of
Addition CPM Control


H20 4213 100

1% DMF 4179 100

Actinomycin D, 100 pg/ml 602 14

Actinomycin D, 75 pg/ml 2052 48

Actinomycin D, 50 pg/ml 4708 100

Pyrophosphate, 2 pmoles/ml 456 10

Phosphate, 2 pmoles/ml 4055 96

Rifampin, 100 Vg/ml 3538 84

Rifamycin AF/013, 100 ug/ml 4050 96

Ethidium Bromide, 100 ug/ml 3370 80

Streptovaricin, 100 Vg/ml 3918 93


The RNA polymerase assay was performed with the addition of
inhibitors to the final concentrations indicated above. Rifam-
pin, rifamycin AF/013, ethidium bromide and streptovaricin were
solubilized in 1% N'N' dimethyl formamide.
















TABLE 7

CHARACTERIZATION OF PRODUCT


Percent
Addition CPM of Control


Control 6412 100

Snake Venom Phosphodiesterase,
10 Pg/ml 561 9

0.1M KOH 1000C, 20 min. 154 2
H20, 1000C, 20 min. 4437 70


0.5M KOH, 370C, 16 hr. 119 2
H20, 370C, 16 hr. 5129 80


The RNA polymerase assay was performed with the addition of
phosphodiesterase (E. C.: 3.1.4.1) from Crotalus atrox venom,
Type VII (Sigma, St. Louis). The alkaline hydrolysis was per-
formed by first performing the RNA polymerase assay for 10
minutes and then treating the reaction mixture with KOH for the
indicated times before the addition of stop bath and the precip-
itation of macromolecules with 10% trichloroachetic acid.










reported by Duda and Cherry (184) for a poly C polymerase activity in

sugar beet nuclei.

The RNA polymerase assay reaction mixture was scaled up so that the

labeled products of the incorporation of 3H-CMP could be studied. For

this purpose the reaction mixture contained in a total volume of 3.0 ml,

5.0 mM of each unlabeled ribonucleoside triphosphate and 0.05 mM of

H-CTP with a specific radioactivity of 2 Ci/mmold. After incubationof

15 min the products were isolated as described in the methods and

material section by phenol extraction followed by ethanol precipitation

of labeled products from the aqueous phase in the presence of carrier

E. coli RNA and CTP. Zonal centrifugation (Figure 4) on sucrose gradient

was performed in order to determine the size of the tritium labeled pro-

duct. The radioactivity remained at the top of the gradient indicating

the material was smaller than that of E. coli 5S RNA. Therefore, the

sizes of the isolated products were estimated by Biogel P-4 column

filtration (Figure 5). The E. coli carrier RNA eluted in the void volume

while two adsorbancy peaks were eluted which contained the labeled cyto-

sine. Using the elution position of a standard CpC the molecular weight

of the first peak (BGP-1) was estimated to be approximately 800 which

corresponds to the molecular weight of a trinucleotide of poly C. The

molecular weight of the second peak (BGP-2) was estimated to be about

680 which corresponds to the molecular weight of a dinucleotide. It

should be noted that this smaller entity elutes before CpC indicating

that it is larger than a dinucleotide of this structure.

The components in the two peaks eluted from the P-4 column have

ultraviolet absorbancy spectrums very similar if not identical to that

of CpC, showing the characteristic absorption maximum and minimum at


















r- E-4 I r-
HC V- c p PQ 4- 1.
*- U ) 0) 0 n
Wn 00 W 0
n 4 n 4 )-
0 U x u m 41 El h0 -

c'H p Q CO


;? W Wo 4 (0' 0
1-1 r 04 0 4J I
o corl > o H I


E (, NZ CO ") 0-O
au-1 -i 400
R u nl g aW V i rVW ^ l -




O) 4J 0) "o 0 0
W0 O CO Wr 0 -U l I



(U CD r H U 0 0- 4 *
oa I-O Ci l M 4.-
Scl U* u

S0 m I 10 o cu4-


U J *H o

S -- I C, r-1 U (0 '
S I C0 0 H i 0 :j O -W
I 0rI 0 00 -
O W CM0 +4-J







0 Z 4iJ MH 0
w- Ca Q) r~ .-i u r i .o
0) i .40 3
0CO r- 4J H 0
0 W 0 d 3 0








N-4 Q) >
H 4-J *H 40) 0-H o
9 M -1 r* M i4J
0 E P JO V 4- 0o


4O W--j >



H w o *- w r. 4J
C)Q rz J tN* Q) r qu
CO 3 C -l Wo
3 *a E 0W i cu rH ni








z H0 WN O Z 0
o W C 0 0 3Ha C) 0)
3 o- I* -j H 0 lV
Z u4 0 P. ,C W








z a P 0 0 0 0
u0 1 4-10 0u-
OH 0) a *J 0 4

ca Qo) U r"l
Sm C *--l h




S o 0) o0
a P 0 > 4hO


U U-*OH OJ0)



S0 4-4 0 1u ro :3
n U 0 Uo m n a w -

I C'i ld 4 l- HO
SU ) 0 4-i 4)- U H0C:
p1 Cl) c 3 r3 'a M 0) 41 4




C *H H -H 0l 40
14 E-4) U W --i CUO 4V 4l 3
HO 0U| C 00 (o. C *H4
























(, 0...)0 9 y v


(-- l X 'W d 3


t*







w










I.
t-






















0- 0




-I

0-l 0





(m *:











l< c
0)











00z o
as












(V-i c> o-





4-1 0.










0 -4
So co
) r -i *















,-I 4
0) >






(0 ai c
e0 *'
Ot *U U

40. Q)
















0 JW0 C

04 l u 4

(0 H




ca 0 0
I .u 4*-i
o O (0 )H
0 4-1 0























0 X0




00 0 *l -
0 *U U



pa m- ii -

P-iZ 0 '
















901 X VJd3
o 0 0
o o 0
1 0






L o
>-



o) N







D 0





0



t.
I(



o0
g /







09 ~---- 1


o09v










pH 2 and pH 7 for cytosine (Figure 6). This demonstrates that cytosine

is the only base incorporated into the products, and suggests that the

products are homopolymers of CMP.

The possibility that the product may be CDP-diglyceride was inves-

tigated, since it has been reported that crude mitochondrial preparations

from rat liver incorporated label from both CTP and dCTP to CDP digly-

ceride and dCTP diglyceride respectively (181). These mitochondria

incorporated label from 3H-dCTP into acid-insoluble material at a much

higher rate than any other deoxyribonucleoside triphosphate. About 95%

-3
of the acid-insoluble material labeled after incubation with 3H-dCTP

could be extracted by chloroform. Therefore, studies were performed to

ascertain if Euglena mitochondria incorporated the label from 3H-CTP into

CDP-diglyceride.

The results of phospholipid extraction are shown in Table 8. A 0.1

ml sample of the 3H-CMP labeled product obtained after nucleic acid

extraction and dissolved in water was mixed with 1.0 ml of methanol for

10 minutes before adding 2.0 ml of chloroform with additional mixing.

Then 4.0 ml of 2M KClwas added with vigorous mixing (Vortex mixer, 5

min., 25 C). After the phases separated upon standing, less than 1% of

the tritium label could be accounted for in the chloroform phase whereas

64% of the label could be accounted for in the aqueous phase. Repeated

additions of the aqueous KC1 to the chloroform phase resulted in no

extraction of additional label into the aqueous phase. The difficulty

in accounting for all of the radioactivity was due to the quencing

produced by the aqueous KC1 phase in the Bray's scintillation fluid.

Further evidence that the product is not CDP-diglyceride was pro-

vided by the failure of the radioactively labeled material eluted from

the P-4 column to migrate in a silica gel G TLC system (Figure 7) in



























bOU

,)0





0
o m



.,j Ct





oa





,C
-4


u

0 -






0 0"


-4 -4
SaW
CU Q















C4



oo



0) (

4-4 bO ct
















I co "'
m P4-4






0 0

40 o U-

-o




O )0

c9 a 0)



D 0







ca
a ac ou)
Mrl 0 0 w












i-I E-I P.






































0
ro
c



-I
N-Z


-J


gONvo0osO V
I .















TABLE 8

PHOSPHOLIPID EXTRACTION OF THE 3H-CMP LABELED PRODUCT


Sample Total CPM % of Control


3H-CTP product 58.50 x 104

First extraction of product
in chloroform solution
Chloroform phase 0.09 x 104 0.2
Aqueous phase 37.20 x 104 64.0

Second extraction of
chloroform phase
Chloroform phase 0.13 x 104 0.2
Aqueous phase 0.09 x 104 0.2

Third extraction of
Chloroform phase
Chloroform phase 0.19 x 10 0.3
Aqueous phase 0.27 x 104 0.4


A 0.1 ml sample of the 3H-CMP-labeled product (containing 58.5 x 104 cpm)
obtained from nucleic acid extraction was dissolved in water and mixed
with 1.0 ml of methonal containing 0.1N HC1. Ten minutes later 2.0 ml
of chloroform was added to the mixture followed by 4 ml of 2M KC1. This
was vigorously mixed on a vortex mixer for 5 minutes at 250. The phases
were allowed to separate upon standing at room temperature and then the
aqueous phase was drawn off and the chloroform phase was extracted 2 more
times. 100 pl samples of each phase were counted for radioactivity as
described in the methods.
















Figure 7. THIN LAYER CHROMATOGRAPHY OF THE 3H-CMP LABELED PRODUCTS
ELUTED FROM THE BIOGEL P-4 COLUMN


Ascending chromatography of the 3H-CMP labeled products eluted from the
Biogel P-4 column was performed according to the method of TerSchegett
et al. (181) on silica gel G in closed tanks at room temperature. The
solvent was chloroform; methanol; water; ammonia (70:30:2:8, v/v). The
standards were C C and CDP-diglyceride. Twenty microliter samples of
the 3H-CMP labeled products and standards were spotted 3.0 cm from the
lower edge of the plate and dried before developing in the solvent. The
800 MW 3H-CMP labeled product (BGP-1) spot contained 35,000 cpm and the
680 MW product (BGP-2) spot contained 35,000 cpm. This was determined
by spotting the same volume of each product on a plate and then removing
a 1.0 cm2 section containing the spot and measuring its radioactivity in
toluene scintillation fluid. The CDP-diglyceride was detected by ultra-
violet light and by staining with iodine vapor. Radioactivity was
detected by sequentially removing 1 cm2 sections from the silica gel plate
and counting them in toluene scintillation fluid.







Silica G
CHLOROFORM: M
AMMONIA
-- SOLVENT FRONT


el G TLC
ETHANOL: WATER:
(70:38:2:8 V/v)


I
Vuv


30200 CPM
35000 CPM
I
1V


21300CPM
24000 CPM

I1n


CpC CDP P-4 COL P-4 COL
DIGLYCER- PEAK I PEAK 2
IDE











which CDP-digylceride migrates (181). Ascending chromatography of the

H-CMP labeled products was carried out as described in the legend.

The labeled products behaved similarly to C C in that they both failed

to migrate in the system and they failed to stain with iodine vapor

indicating that they contained no lipid moieties. As noted in Figure 7

100% of the cmp were recovered in the 1 cm square section comprising the

origin. No significant radioactivity was detected in the other 1 cm

squares which were sequentially removed from the plate and counted for

radioactivity. The CDP-diglyceride standard migrated in the system and

stained with iodine indicating that the radioactive products are not

CDP-diglyceride.

Attempts were then made to ascertain the structure of the labeled

products. This involved treating materials eluted from the P-4 column

peaks with nucleases and analyzing the cleavage products by their migra-

tion in a PEI cellulose TLC system in which ribonucleoside monophosphates

can be distinguished. Treatment of the labeled material (BGP-1 and

BGP-2) eluted from the P-4 column peaks with snake venom phosphodiesterase

resulted in 93% of the radioactive label migrating to a position correspon-

ding to 5'-CMP (Figure 8 and 9), the expected cleavage products of poly C

and CpC. However, when the products were treated with RNase A, the radio-

active label remained at the origin as did the untreated products. This

data indicates that the products were insensitive to RNase A. This being

the case, then the products have a chemical structure which is different

from that of a homopolymer of CMP since RNase A cleaves CpC to the expected

2', 3' CMP products. However, the possibility exists that these were

homopolymers which have modified 3' terminus which prevents the RNase A

from binding. The possibility of a small oligonucleotide with a 5'
















Figure 8. THIN LAYER CHROMATOGRAPHY OF THE PRODUCTS OF THE ENZYMATIC
CLEAVAGE OF THE 3H-CMP LABELED MATERIAL ELUTED FROM THE P-4
COLUMN


Ascending chromatography of the products of the enzymatic cleavage of
the 3H-CMP labeled material eluted from the P-4 column was performed
on PEI cellulose as described in the methods. The molecules eluted
from the Biogel P-4 column BGP-1 (800 MW) and BGP-2 (680 MW) were
treated separately with each of the digestive enzymes; snake venom
phosphodiesterase, and ribonuclease A. The standards were CpC, 2'-
3'-CMP, and 5'CMP. Twenty microliters of each digest was spotted 3.0
cm from the bottom of the plate and dried. The enzymatic digest spots
contained the cpm indicated in the denominators. Ribonucleoside mono-
phosphates were chromatographed as described in the methods. The
nucleotides were detected with ultraviolet light. Radioactivity was
detected by sequentially removing 1.5 cm2 sections of the PEI cellulose
and counting them in toluene scintillation fluid.












PEI Cellulose TLC
LON ACETIC ACID TO 4CM
0.3 LiC to 15 CM
SOLVENT FRONT


5456 CPM
5839 CPM
D


QUV


Quv (


5196 CPM
6954 CPM

D


8170 CPM
8636 CPM
[ I.. .


P-4 COL P-4 COL P-4 COL CpC CpC 2', 3'-CMP 5'-CMP
UN- PEAKS PEAKS + +
+ +
TREATED S.VR RNaseA S.VP RNaseA


I
















Figure 9.


THE MIGRATION OF THE ENZYMATIC CLEAVAGE PRODUCTS CONTAINING
TRITIUM LABEL ON PEI CELLULOSE


The migration of the enzymatic cleavage products containing tritium
label on PEI cellulose as described in Figure 8.


























































DISTANCE (cm)


m










blocked terminus and/or methylated nucleoside moieties can not be ruled

out.


The Solubilization and Partial Purification of
Euglena Mitochondrial RNA Polymerase

The final objectives were to solubilize and purify the mitochondrial

DNA-directed RNA polymerase, to characterize its activity, to determine

its requirements for product synthesis, and to compare it to nuclear RNA

polymerase activities with respect to these requirements. In order to

accomplish this it was necessary to prepare mitochondria which were devoid

of contaminating nuclear DNA. This was achieved by obtaining a crude 5P

mitochondrial pellet, as described in Figure 1, and washing it twice in

STM Buffer before incubating the mitochondria in the presence of deoxyri-

bionuclease I (DNase I) for thirty minutes, zero degrees centigrade.

These mitochondria were shown to be devoid of nuclear DNA by work substan-

tiated in this laboratory and no further purification was necessary. The

buoyant densities of the DNA in the mitochondrial preparations in CsC1 are

demonstrated in Figure 10. The crude washed 5P mitochondria yield mostly

Euglena nuclear DNA (Tube a) which has a density of 1.707 g/cm but after

five minutes of incubation in DNase I at 4 OC, most of the nuclear DNA was

removed (Tube b). Thirty minutes of incubation in DNase removes all of

the contaminating nuclear DNA and only mitochondrial DNA remains, which

has a density of 1.691 g/cm3 (Tube c). Further treatment with DNase for

one hour shows that the mitochondrial DNA is still present and is there-

fore protected from digestion by DNase I by the mitochondrial membranes.

The mitochondria purified in this manner demonstrated the same ribonucle-

otide incorporating activities as the mitochondria purified on renografin

gradients. Table 9 shows that the DNased treated mitochondria incorpora-



















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TABLE 9


A COMPARISON OF THE INCORPORATION OF 3H-UTP
ISOLATED MITOCHONDRIA PURIFIED BY TREATMENT


and 3H-CTP BY
WITH DNASE I


Labeled Reaction
Ribonucleotide Mixture CPM


3H-UTP Complete 1,624

3H-UTP -DNA 869

H-CTP Complete 27,364

3H-CTP -DNA 16,669


Mitochondria purified by DNase I treatment were treated with isotonic
buffer to assure swelling and permability. The RNA polymerase assay
was performed as described in the methods. Reaction mixtures of 0.2
ml contained 1.0 mM of each unlabeled NTP and 0.016 mM 3H-UTP or 3H-CTP,
2.Ci/mmole.










ted tridum from H-CTP at a ten fold greater rate than H-UTP. However,

about fifty percent of the incorporating activities from both 3H-CTP and

3H-UTP are DNA dependent which is in contrast to the 3H-CTP incorporating

activities in renografin purified mitochondria which was not DNA dependent.


Solubilization of the Mitochondrial
RNA Polymerase Activity

The difficulty of defining proper conditions for solubilization of

the chloroplast RNA polymerase of Euglena gracilis (166,185) plus the

rapid inactivation of mitochondrial RNA polymerase activities by some

solubilization methods (11), prompted the investigation to determine

optimum conditions for solubilizing the activity from Euglena mitochon-

dria (Table 10). An approach used by Rogall and Wintersherger (15) were

used to determine the optimum conditions for solubilization of the

mitochondrial RNA polymerase. A volume of mitochondrial suspension in

lysis buffer was added to an equal volume of the detergent-KCl solution

to bring the mixture to the indicated final concentrations. The mixtures

were centrifuged at 30,000 rpm to sediment non-solubilized material and

the supernatants were assayed for RNA polymerase activities. The

efficiencies of the different treatments in the solubilizations of RNA

polymerase activity from the mitochondria are summarized in Table 10.

It can be seen that the combined treatment with KC1 and Triton X-100

produced the highest yield of activity solubilized. However, NP-40, a

detergent employed by other investigators (15,173), and 0.5 M KC1 was

almost as effective. Treatment with 0.5 M KC1 alone failed to solubi-

lize significant RNA polymerase activity. Treatment with either deter-

gent alone was significantly less than with the Triton X-100 plus 0.5

M KC1 treatment which completely destroyed the succinic dehydrogenase
















TABLE 10

RNA POLYMERASE ACTIVITY SOLUBILIZED FROM
MITOCHONDRIA


Treatment of RNA Polymerase Activity
Mitochondria (units/g wet wt.)
in Lysis Buffer
30KS 30KP

+ Water 4.2 69

+ 0.5 M KC1 4.2 63

+ 0.8% NP-40 45.5 46

+ 0.8% Triton X-100 34.0 37

+ 0.8% NP-40 + 0.5 M KC1 56.0 0

+ 0.8% Triton + 0.5 M KC1 68.0 15

+ Water (Sonication) 0 28


Mitochondria purified by DNase treatment were suspended in lysis buffer
(Ig wet wt/10 ml). Mitochondrial samples of 1.0 ml each were then added
to an equal volume of the indicated lysis mixture which contained 0.01 M
EDTA plus additions shown. The mixtures were mixed gently for 15 min. at
40C and then centrifuged for 90 min. at 30,000 rpm (Ti 50 rotor). The
supernatants and pellets were dialyzed against TGMED Buffer for 4 hrs.
before analyzing them for RNA polymerase activity as described in the
methods. The concentration of 3H-UTP was 0.016 mM with a specific radio-
activity of 2 Ci/mmole.










activity (Table 11) in the supernatant. The NP-40 plus 0.5 M KC1 treat-

ment produced supernatants that contained succinic dehydrogenase activi-

ties as high as they were in the control mitochondrial pellet. This

suggested that Triton X-100 detergent was effective in completely

disrupting the inner membrane thus freeing the succinic dehydrogenase of

the membrane associated lipids which are needed to reconstitute the

activity. The NP-40 detergent does not seem to be able to free the suc-

cinic dehydrogenase enzyme from the associated lipid since any loss of

activity in the pellet could be observed as activity in the supernate

indicating membrane components are present in this fraction needed for the

expression of succinic dehydrogenase activity. The NP-40 detergent even

with 0.5 M KC1 does not seem to be able to free the succinic dehydrogenase

enzyme from the associated membrane components. It appears that although

there is 80% as much RNA polymerase activity in the 30,000 rpm supernatant

produced by NP-40 as there is in the triton treated supernatant, the NP-40

supernatant may not contain solubilized RNA polymerase but just small

mitochondrial fragments that fail to sediment at 30,000 rpm in 90 minutes.

Therefore, the 0.8% Triton X-100 treatment with 0.5 M KC1 was used for

solubilizing the mitochondrial RNA polymerase.


The Effect of Ammonium Sulfate Precipitation on
Mitochondrial RNA Polymerase Activity

It has been reported that Neurospora mitochondrial RNA polymerase

(11) activity could not survive ammonium sulfate precipitation, chroma-

tography on DEAE-Cellulose, extensive dialysis, concentration by

ultrafiltration, and sucrose gradient centrifugation. Therefore, it was

necessary to determine if Euglena mitochondrial RNA polymerase could

survive ammonium sulfate treatment because the selection of proper salt
















TABLE 11

SUCCINIC DEHYDROGENASE ACTIVITY IN THE MITOCHONDRIAL
SUPERNATANTS AND PELLETS AFTER SOLUBILIZATION
TREATMENT


Treatment of Succinic Dehydrogenase Activity
Mitochondria (Units x 10-3)
in Lysis Buffer 30KS 30KP

+ Water 0 40

+ 0.5 M KC1 0 49

+ 0.8% NP-40 22 27

+ 0.8% Triton X-100 0 31

+ 0.8% NP-40 + 0.5 M KC1 34 0

+ 0.8% Triton + 0.5 M KC1 0 0

+ Water (sonication) 0 0


A unit is a pmole of dichlorophenolindophenol reduced per minute per
ml of enzyme sample at 250C.










concentration would allow the fractionation of RNA polymerase away from

other mitochondrial proteins, thus effecting another purification step.

The effect of ammonium sulfate on the mitochondrial RNA synthesizing

activity was measured (Table 12) by adding to solubilized mitochondrial

RNA polymerase samples ammonium sulfate to the desired concentration with

mixing followed by centrifugation at 40,000 rpm for 90 minutes. The RNA

polymerase activity was measured in the redissolved and dialysed protein

samples as described in the methods. The results indicated that 90% of

the activity was recovered when the ammonium sulfate concentration was

50%. It was interesting to observe that when the 30,000 rpm super

(control) was centrifuged at 40,000 rpm for 70 min. 19% of the activity

was lost. This could represent the activity that is not completely

solubilized and thus sediments at 40,000 rpm but not at 30,000 rpm.


Solubilization and Purification
Of Mitochondrial RNA Polymerase

The scheme for the purification of the mitochondrial RNA polymerase

is shown in Figure 11. The optimum conditions described in Table 10

were used to solubilize mitochondrial RNA polymerase. The washed mito-

chondria obtained from 182g (wet weight) of cells were purified by

treatment with pancreatic deoxyribonuclease (100 pg/ml) for thirty minutes.

The mitochondria obtained after two washings in STE buffer were suspended

in lysis buffer and either used directly for enzyme solubilization or

stored in liquid nitrogen prior to use. An equal volume of lysis mixture

was mixed with mitochondrial suspension to bring the final concentration

to 0.8% Triton X-100, 0.50 M KC1, and 0.005M EDTA. The mixture was

gently stirred for 15 minutes, 4C. The supernatant obtained after cen-

trifuging at 30,000 rpm for 90 minutes was brought to 50% saturation with















TABLE 12

THE EFFECT OF AMMONIUM SULFATE PRECIPITATION ON MITOCHONDRIAL
RNA POLYMERASE ACTIVITY


%
Treatment % (NH4)2SO4 Activity Recovered
Saturation (CPM/ml) in Pellet

30K Super 12420

+ 0, 40KP 1915 19

+ 30, 40KP 5660 46

+ 40, 40KP 6640 53

+ 50, 40KP 11220 90

+ 60, 40KP 6220 50

+ 65, 40KP 8830 71

+ 70, 40KP 9880 80

+ 75, 40KP 9090 73

+ 80, 40KP 7520 61


Mitochondria purified by DNase treatment were suspended in lysis
buffer (1 g wet wt/10 ml) and the RNA polymerase activity was
solubilized as described in the methods. Five milliliters of the
mitochondrial suspension were added to an equal volume of the lysis
mixture (1.6% Triton X-100, 1.0 M KC1, and 0.01 M EDTA pH 7.9) and
mixed gently for 15 min. at 40C and then centrifuged for 90 min. at
30,000 rpm (Ti 50 rotor). The supernatants were divided into 1.0
ml aliquots and solid ammonium sulfate was added to each aliquot to
the desired concentration. The ammonium sulfate was dissolved at
40C by gently mixing and the precipitated protein was collected by
centrifugation at 40,000 rpm, 70 min. (Ti 50 rotor). Each pellet was
dissolved in 1.0 ml of TGMED buffer and dialyzed against 2 liters of
TGMED for 4 hrs. at 40C. The ammonium sulfate concentration in each
dialysate was determined with the conductance bridge and the salt
concentration was adjusted to 0.05 M ammonium sulfate. The RNA
polymerase assay was performed as described in the methods except that
KC1 was omitted.










Washed 5P Mitochondria
DNased, 30 min, 40C
Rx Stopped w/3 vol. 0.1 M EDTA pH 7.5: 10% Sucrose

cent. 10,000 x g, 20 min (Repeat 2X in STE buffer)


Purified Mitochondria

Resuspended in lysis buffer
Add equal vol. lysis mixture:
1.6% Triton X-100
1.0 M KC1
0.01 M EDTA

Cent. 30,000 rpm, 90 min, 20C


30 KP


50% (NH4)2SO4 situation

Cent. 40,000 rpm, 70 min, 20C



40 KP


Dissolved in TGMED Buffer

Dialyze 4 hr against 2 liters
TGMED: 40 mM (NH )2SO4

DEAE-Sephadex A-25
column chromatography


DCI


Figure 11.


PROCEDURE FOR THE PARTIAL PURIFICATION OF MITOCHONDRIA
RNA POLYMERASE


10 KS


30 KS


40 KS










respect to ammonium sulfate. The precipitation collected at 40,000 rpm

was dissolved in TGMED buffer and dialysed against TGMED containing 50 mM

ammonium sulfate. The dialysate was subjected to DEAE-Sephadex chroma-

tography and the eluted fractions were assayed for 3H-CTP incorporating

activities using the RNA polymerase assay (Figure 12). The void volume

contained an activity which incorporated tritium preferrentially from

the substrate 3H-CTP by a reaction which was not dependent upon added

DNA or the other Ribonucleotide substrates into acid insoluble products

which resisted pancreatic ribonuclease digestion (Table 13). However,

an activity which incorporated label from 3H-UTP into acid insoluble and

RNase digestible products was eluted from the DEAE-Sephadex column

(Figure 12) in a single peak between 0.32M and 0.37M ammonium sulfate in

TGMED buffer. The specific activity of the RNA polymerase after this

purification step was 0.3 nmoles UMP incorporated/mg protein (estimated

from A280 absorbancy)/10 minutes at 37 C. This enzyme activity demon-

strates a dependence upon added DNA and ribonucleoside triphosphate

substrates (Table 14) for the incorporation of label from 3H-UTP. The

mitochondrial RNA polymerase activity is also inhibited by low concentra-

tions (10 ug/ml) of actinomycin D (Table 15). The activity is insensi-

tive to rifampin, a-amanitin, streptovaricin, rifamycin AF/013 and

rifamycin AF/05. Attempts to concentrate this activity by rechromato-

graphy on a smaller DEAE-Sephadex A-25 column or to further purify the

activity by glycerol gradient centrifugation resulted in the loss of

significant activity.






















C) 3 U





Ia a)



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xa) 4







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U) C u



W -H d
I <

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< *H -




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u *Hr Q) H







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0 0 c




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TABLE 13

A COMPARISON OF TRITIUM INCORPORATION FROM 3H-UTP and 3H-CTP
BY THE ACTIVITY IN THE DEAE-SEPHADEX A-25 COLUMN FRACTION 3


Reaction
Mixture CPM


3H-UTP
Complete 200

3H-CTP
Complete 5428
-DNA 2581
-NTP 4389
+RNase 5316


The RNA polymerase assay reaction mixtures contained 95
pl of the void volume activity in a total volume of 0.20
ml. The concentration of 3H-CTP, were 0.016 mM, 2 Ci/mmole.
















TABLE 14

THE DEPENDENCY OF THE DEAE COLUMN FRACTION 38 INCORPORATION
OF 3H-UMP UPON ADDED RIBONUCLEOSIDE TRIPHOSPHATES AND DNA


Additions Percent
or of
Deletions CMP Control

Complete 1488

-DNA 187 13

-NTP 398 27

-ATP 168 11

-CTP 238 16

-GTP 178 12

+ Pancreatic RNase
(100 gg/ml) 355 24


of DCI
was 0.016


The RNA polymerase assay reaction mixtures contained 20 pl
in a total volume of 0.20 ml. The concentration of 3H-UTP
mM, 2 Ci/mmole.












TABLE 15


INHIBITORS OF H-UTP
COLUMN FRACTION 38


INCORPORATION BY THE DEAE


Inhibitors CPM % Activity


Distilled Water 2496 100

1% DMF 2521 100

Actinomycin D, 10 ug/ml 82 3

Rifampin, 100 pg/ml 1898 76

Rifamycin AF/013, 100 ug/ml 2109 85

Rifamycin AF/05, 100 ug/ml 2065 83

Streptovaricin, 100 yg/ml 2369 95

a-Amanitin, 5 ug/ml 1490 60

Pancreatic RNase 100 pg/ml 69 3


The RNA polymerase assay was performed with the addition of
inhibitors to the final concentrations indicated above. The
concentration of 3H-UTP was 0.016 mM, 2 Ci/mmole. Rifampin,
rifamycin AF/013, rafamycin AF/05, and streptovaricin were sol-
ubilized in 1% N'N' dimethyl formamide. Reaction mixtures con-
tained 50 ml of the DCI enzyme in a total volume of 0.2 ml.










Enzymatic Properties of the
Mitochondrial RNA Polymerase

The dependence of the mitochondrial RNA polymerase on the concen-

tration of MnC12 is shown in Figure 13. Low concentration (about 1 mM)

of Mn+ stimulated the activity of this enzyme and higher concentrations

decreased the activity. However, the enzyme activities were also stimu-

lated by low concentrations of Mg (Figure 14) demonstrating the optimal

concentration at 0.5 mM.

The template requirement of the mitochondrial enzyme is summarized

in Table 16. The mitochondrial enzyme prefers poly d(AT) as a template

which is similar to the rifampicin sensitive mitochondrial RNA polymerase

from Neurospora crassa, described by Kuntzel and Schafer (11). It is

interesting to note that while Euglena nuclear DNA was a more effective

template than denatured calf thymus DNA that it is not necessarily a

more preferred template than Euglena mitochondrial DNA. The mitochondrial

DNA was only available in 1/5 the saturating DNA concentration and a

comparison of the templates was not possible.
















Figure 13.


DEPENDENCE OF THE ACTIVITY OF THE DEAE FRACTION 38
ON THE CONCENTRATION OF Mn++


Assays were carried out as described in methods. The concentration
of 3H-UTP was 0.016 mM, 2 Ci/mmole. The reaction mixtures contained
20 pl of DCI enzyme in a total volume of 0.2 ml.



















CPM


mM Mn"
















Figure 14.


DEPENDENCE OF THE ACTIVITY OF THE DEAE FRACTION 38
ON THE CONCENTRATION OF Mg++


Assays were carried out as described in methods. The concentration of
3H--UTP was 0.016 mM, 2 Ci/mmole. The reaction mixtures contained 20 ip
of DCI enzyme in a total volume of 0.2 ml.