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Role of A18 in Vaccinia Virus Post-replicative Gene Transcription Termination

Permanent Link: http://ufdc.ufl.edu/UFE0042065/00001

Material Information

Title: Role of A18 in Vaccinia Virus Post-replicative Gene Transcription Termination
Physical Description: 1 online resource (124 p.)
Language: english
Creator: Manoharan, Aparna
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: a18, post, termination, transcription, vaccinia
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Role of A18 in Vaccinia Virus Post-replicative Gene Transcription Termination Vaccinia virus is a large double stranded DNA virus, unique among DNA viruses because it replicates entirely in the host cytoplasm and hence encodes for its own transcriptional machinery. Elucidation of the process of transcription in vaccinia has served as an excellent model to understand different aspects of eukaryotic transcription biology. Transcription in vaccinia virus is temporally regulated, with the genome being transcribed as early, intermediate and late genes. Intermediate and late gene expressions require the onset of genome replication and hence are referred to as post-replicative gene expression. Our lab is specifically interested in the process of transcription regulation of post-replicative genes in vaccinia virus. Several viral proteins have been identified to play a role in post-replicative transcription regulation. This dissertation is based on work done with one such protein, A18, and its role in viral transcription termination. The vaccinia virus (VV) protein A18 was identified in our lab to play a role in late gene transcription regulation, from studies done with temperature sensitive mutant viruses. The mutant viruses made late transcripts that were longer than wild-type virus transcripts, implying that the mutant gene product, A18, functioned as a negative elongation factor. Purified histidine-tagged A18 shows DNA-dependent ATPase activity and a weak helicase activity for dsDNA. An in vitro transcription assay shows that A18 functions as a transcript release factor in the presence of additional host factors from uninfected HeLa extracts. This dissertation involves studying A18 in the context of an in vitro transcription assay to understand the role of A18 as a transcription termination factor. Attempts made to characterize the nature of the host factor indicated that a specific, ubiquitous eukaryotic protein component of the HeLa extracts aided in A18 mediated termination. Stalled transcription ternary complexes generated from a vaccinia intermediate gene promoter that were isolated and used to study A18 mediated termination showed that neither A18 nor the host factor were required to interact with the elongating RNA polymerase complex in order to mediate termination. These stalled ternary complexes proved an important tool to delve into the nature of A18 mediated termination and study the reaction kinetics, define salt and divalent metal ion optima and energy requirements for this enzymatic process. An important aspect of the in vitro studies with A18 involves looking at the role of the enzyme in the context of transcriptional pausing. We have been able to show that the enzyme mediates termination of ternary complexes paused at natural pause sites, proving that pausing is a requirement for A18 mediated termination. In the context of transcriptional pausing, we have also identified that A18 has to a certain degree host factor independent termination potential.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Aparna Manoharan.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Condit, Richard C.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042065:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042065/00001

Material Information

Title: Role of A18 in Vaccinia Virus Post-replicative Gene Transcription Termination
Physical Description: 1 online resource (124 p.)
Language: english
Creator: Manoharan, Aparna
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: a18, post, termination, transcription, vaccinia
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Role of A18 in Vaccinia Virus Post-replicative Gene Transcription Termination Vaccinia virus is a large double stranded DNA virus, unique among DNA viruses because it replicates entirely in the host cytoplasm and hence encodes for its own transcriptional machinery. Elucidation of the process of transcription in vaccinia has served as an excellent model to understand different aspects of eukaryotic transcription biology. Transcription in vaccinia virus is temporally regulated, with the genome being transcribed as early, intermediate and late genes. Intermediate and late gene expressions require the onset of genome replication and hence are referred to as post-replicative gene expression. Our lab is specifically interested in the process of transcription regulation of post-replicative genes in vaccinia virus. Several viral proteins have been identified to play a role in post-replicative transcription regulation. This dissertation is based on work done with one such protein, A18, and its role in viral transcription termination. The vaccinia virus (VV) protein A18 was identified in our lab to play a role in late gene transcription regulation, from studies done with temperature sensitive mutant viruses. The mutant viruses made late transcripts that were longer than wild-type virus transcripts, implying that the mutant gene product, A18, functioned as a negative elongation factor. Purified histidine-tagged A18 shows DNA-dependent ATPase activity and a weak helicase activity for dsDNA. An in vitro transcription assay shows that A18 functions as a transcript release factor in the presence of additional host factors from uninfected HeLa extracts. This dissertation involves studying A18 in the context of an in vitro transcription assay to understand the role of A18 as a transcription termination factor. Attempts made to characterize the nature of the host factor indicated that a specific, ubiquitous eukaryotic protein component of the HeLa extracts aided in A18 mediated termination. Stalled transcription ternary complexes generated from a vaccinia intermediate gene promoter that were isolated and used to study A18 mediated termination showed that neither A18 nor the host factor were required to interact with the elongating RNA polymerase complex in order to mediate termination. These stalled ternary complexes proved an important tool to delve into the nature of A18 mediated termination and study the reaction kinetics, define salt and divalent metal ion optima and energy requirements for this enzymatic process. An important aspect of the in vitro studies with A18 involves looking at the role of the enzyme in the context of transcriptional pausing. We have been able to show that the enzyme mediates termination of ternary complexes paused at natural pause sites, proving that pausing is a requirement for A18 mediated termination. In the context of transcriptional pausing, we have also identified that A18 has to a certain degree host factor independent termination potential.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Aparna Manoharan.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Condit, Richard C.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042065:00001


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Full Text

PAGE 5

E.coli In Vitro

PAGE 6

In Vitro In Vitro

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in vitro in vitro in vitro

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Transcription & Gene Expression

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Prokaryotic Transcription E.coli

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RNA P olymerase E.coli

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Initiation in vitro

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Elongation

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Termination Rho dependent termination E.coli in vitro

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in vitro

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Intrinsic termination In vitro

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Mfd mediated termination

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in vitro Eukaryotic Transcription

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RNA P olymerase

PAGE 22

Initiation

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Elongation

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Termination

PAGE 29

in vitro in vitro

PAGE 31

in vitro in vitro in vitro in vitro

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Vaccinia Virus Biology

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Vaccinia Life Cycle

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Vaccinia Virus T ranscription

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in vitro

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In vitro

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Early gene expression

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in vitro In vitro

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Post replicative gene expression

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in vitro

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In vitro in vivo In vitro

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in vitro A18 in vitro

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In vivo In vivo in vitro in vitro in vivo Significance of this S tudy

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in vitro in vitro

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Eukaryotic Cells, Prokaryotic Hosts, Viruses and E xtracts Plasmids in vitro

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Tra nscription Competent Lysolecithin E xtracts HeLa Cytoplasmic Extracts

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Preparation of His A18 E.coli Lysate Preparation

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Purification on a HisTrap C olumn Templates Used in In Vitro Transcription A ssays Preparation of pG8G Template

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Preparation of pG8GU Template In Vitro T ranscripti on Release of Stalled T ernary C omplexes

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In Vitro T ranscriptio n Release of Paused Elongation C omplexes

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in vivo in vitro in vitro in vitro Specific Aim 1: Is A18 Mediated T e rminati on Functionally Dependent on E longation? in vitro in vitro

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Iso lation of the Stalled Ternary C omplex in vitro

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Are A18 and the Host Extracts Required During Elongation In Order to Mediate T ermination?

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Specific Aim 2: Characterization of the Host Factor A ctivity in vitro in vitro

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Non specific Substitution of Host Factor A ctivity in vitro in vitro in vitro

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Deterg ent Stimulation of A18 mediated T ermination in vitro

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Heat S tability of t he Host Factor A ctivity Host Factor is a P rotein

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Host Factor is a Ubiquitous Eukaryotic P rotein in vitro

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Sp e cific Aim 3: Properties of A18 M ediated Termination of Stalled Ternary C omplexes Kinetics of T ermination of STC

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Salt Optima for T ermination

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Divalent Metal Ion Optima for T ermination

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Energy Requirement for Termination in vitro

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Specific Aim 4: A18 Mediated Termination of Paused C omplexes

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Termination of Paused E longation C omplexes

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Early Time Course of E longation

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A1 8-s pecific Termin ation of Paused C omplexes

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in vitro H ost Factor Requirement for A18 Mediated Termination

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in vitro Properties of A18 M ediated Termination of STC

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in vitro in vitro A18 M e diated Termination and Transcriptional Pausing

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in vivo Model for A18 Mediated Termination

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in vitro

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in vitro

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in vitro Role of A18 in Viral Post replicative Transcription Regulation

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in v itro in vitro

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Future Directions

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in vitro in vitro

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in vitro in vitro

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in vitro

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In vitro in vitro

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in vitro

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Escherichia coli In vivo in vitro

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in vitro Saccharomy ces cerevisiae in vivo Escherichia coli Escherichia coli

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Saccharomyces cerevisiae Escherichia coli Escherichia coli in vivo in vitro E. coli

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Escherichia coli In vitro Escherichia coli

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E. coli

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Escherichia coli Escherichia coli in vitro E. coli

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in vitro Saccharomyces cerevisiae Escherich ia coli Escherichia coli Escherichia coli

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Escherichia coli in vitro

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Thermus aquaticus