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Sequence-specific interactions between RNA polymerase and the core recognition element

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Title
Sequence-specific interactions between RNA polymerase and the core recognition element
Name (type = personal)
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Vahedian Movahed
NamePart (type = given)
Hanif
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Hanif Vahedian Movahed
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author
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Ebright
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Richard H.
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Richard H. Ebright
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Advisory Committee
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chair
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Bryce E
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co-chair
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Arnold
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Eddy
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Eddy Arnold
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Advisory Committee
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internal member
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Patel
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Smita
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Smita Patel
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internal member
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Olson
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Wilma
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Wilma Olson
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Advisory Committee
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outside member
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Rutgers University
Role
RoleTerm (authority = RULIB)
degree grantor
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NamePart
School of Graduate Studies
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school
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Text
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theses
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DateCreated (qualifier = exact)
2017
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2017-10
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2017
Place
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xx
Language
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eng
Abstract (type = abstract)
In bacteria, the flow of biological information from DNA to RNA is carried out by a single enzyme called RNA polymerase (RNAP). Bacterial RNAP is composed of a multi-subunit catalytic core and a dissociable subunit called sigma factor. Since the discovery of sigma factor in 1969, the prevailing view has been that the RNAP core enzyme requires binding to sigma for sequence-specific transcription, because the RNAP core does not contain the determinants for sequence-specific core promoter recognition and DNA unwinding. It has also been assumed that sequence-specific RNAP-DNA interactions are mainly limited to transcription initiation, as sigma could dissociate from the RNAP core after the initiation stage. These two paradigmatic assumptions have been challenged by recent structural evidence from our lab, which indicates in the initiation complex, the RNAP core directly interacts with the non-template strand segment of the transcription bubble corresponding to positions -4 to +2, and that the interaction with this element is sequence-specific at least at one of its positions. This element has been termed the “core recognition element,” CRE. This thesis addresses three major topics regarding CRE: sequence-specificity, recognition mechanism, and the functional roles.

In chapter 1, using equilibrium binding and dissociation kinetics studies, I demonstrate that the RNAP core shows sequence-specificity at 3-out-of-6 CRE positions (the consensus sequence is T₋₄ n₋₃ n₋₂ n₋₁ T₊₁ G₊₂). I also determine that RNAP amino acid βR371 mediates specificity at CRE position -4, βW183 mediates specificity at CRE position +1, and βR151, βD446, or βR451 mediates specificity at CRE position +2. In subsequent chapters, I use the RNAP derivative containing the βD446A substitution as a reagent to assess the functional significance of RNAP-CRE⁺²ᴳ interactions on transcription initiation and elongation.

In chapters 2, 3 and 4, using a combination of next-generation sequencing approaches and biophysical and biochemical assays, I show that sequence-specific RNAP-GCRE interactions play functional roles in three key stages of transcription initiation: promoting DNA unwinding at a consensus GCRE sequence, favoring start-site selection at positions upstream of a consensus GCRE sequence, and reducing the probability of abortive transcript release at positions upstream of a consensus GCRE sequence.

In chapter 5, using biochemical assays and mNET-seq, I show that sequence-specific RNAP-GCRE interaction occurs in and plays functional roles in key stages of transcription elongation through the E. coli genome: favoring pause-read-through at positions upstream of consensus GCRE sequence and favoring post-translocated states at positions upstream of consensus GCRE sequence.

In chapter 6, using a promoter-independent transcription assay, I show that RNAP-GCRE interaction occurs in, and plays functional roles in all three domains of life: bacteria, archaea and eukaryote.

In chapter 7, using genome-wide next-generation sequencing approaches, I show that the RNAP core can perform sequence-specific transcription in the absence of sigma factor in a manner that correlates with the presence of an AT-rich region followed by a TG-motif.

In the final chapter, I summarize how my work revealed previously undocumented regulatory events in transcription initiation and elongation. Based on my findings, I describe two implications of this thesis for future consideration: a scenario describing what the architecture of primordial promoter sequences might have looked like and a mechanism for antibiotic-tolerant persistence state.
Subject (authority = RUETD)
Topic
Biochemistry
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Title
Rutgers University Electronic Theses and Dissertations
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ETD_8444
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electronic resource
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application/pdf
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text/xml
Extent
1 online resource (xvi, 163 p. : ill.)
Note (type = degree)
Ph.D.
Note (type = bibliography)
Includes bibliographical references
Subject (authority = ETD-LCSH)
Topic
RNA polymerases
Note (type = statement of responsibility)
by Hanif Vahedian-Movahed
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Title
School of Graduate Studies Electronic Theses and Dissertations
Identifier (type = local)
rucore10001600001
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NjNbRU
Identifier (type = doi)
doi:10.7282/T38055RD
Genre (authority = ExL-Esploro)
ETD doctoral
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The author owns the copyright to this work.
RightsHolder (type = personal)
Name
FamilyName
Vahedian Movahed
GivenName
Hanif
Role
Copyright Holder
RightsEvent
Type
Permission or license
DateTime (encoding = w3cdtf); (qualifier = exact); (point = start)
2017-09-29 03:11:02
AssociatedEntity
Name
Hanif Vahedian Movahed
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Copyright holder
Affiliation
Rutgers University. School of Graduate Studies
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Author Agreement License
Detail
I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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DateTime (encoding = w3cdtf); (qualifier = exact); (point = start)
2017-10-31
DateTime (encoding = w3cdtf); (qualifier = exact); (point = end)
2019-10-31
Type
Embargo
Detail
Access to this PDF has been restricted at the author's request. It will be publicly available after October 31st, 2019.
Copyright
Status
Copyright protected
Availability
Status
Open
Reason
Permission or license
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