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The use of single-molecule DNA nanomanipulation to study transcription kinetics

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Title
The use of single-molecule DNA nanomanipulation to study transcription kinetics
Name (ID = NAME001); (type = personal)
NamePart (type = family)
Liu
NamePart (type = given)
Chen-Yu
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Chen-Yu Liu
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author
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Ebright
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Richard
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Advisory Committee
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Richard H. Ebright
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chair
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NamePart (type = family)
Levy
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Ronald
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Advisory Committee
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Ronald M. Levy
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internal member
Name (ID = NAME004); (type = personal)
NamePart (type = family)
Olson
NamePart (type = given)
Wilma
Affiliation
Advisory Committee
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Wilma K. Olson
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internal member
Name (ID = NAME005); (type = personal)
NamePart (type = family)
Ludescher
NamePart (type = given)
Richard
Affiliation
Advisory Committee
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Richard D. Ludescher
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outside member
Name (ID = NAME006); (type = corporate)
NamePart
Rutgers University
Role
RoleTerm (authority = RULIB)
degree grantor
Name (ID = NAME007); (type = corporate)
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Graduate School - New Brunswick
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school
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Text
Genre (authority = marcgt)
theses
OriginInfo
DateCreated (qualifier = exact)
2007
DateOther (qualifier = exact); (type = degree)
2007
Language
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English
PhysicalDescription
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electronic
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application/pdf
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xvi, 145 pages
Abstract
Transcription involves many reaction steps and intermediates. Many phenomena in transcription kinetics are covered by ensemble average. Single-molecule DNA nanomanipulation techniques uncover these transcription kinetic events via determination of a transcription bubble in real time. In this dissertation, we focus on the transcription kinetics of bacterial RNAP. The study of transcription kinetics in this thesis can be divided into 4 main subjects: 1) The study of abortive initiation mechanism: Through a single-molecule DNA nanomanipulation technique, we tested the three models proposed for the mechanism of abortive initiation - inchworming, scrunching and transient excursion - on T5 N25 promoter. Of the three models, only the scrunching model involves a change in the size of the transcription bubble during abortive initiation, which was observed by single-molecule DNA nanomanipulation technique. 2) The study of the kinetics of elongation and termination: By introducing varying transcribed region lengths into DNA templates, the kinetics of elongation and terminator rewinding were studied. The resulting kinetics, determined by the single-molecule nanomanipulation technique, was analyzed by different regression methods. In the normal regression, the elongation velocity is 10 b/s and terminator rewinding takes 3.4 s on a tR2 terminator. Contrarily, through Poisson regression, the elongation velocity ranges from 6.4 b/s to 12.5 b/s and terminator rewinding takes 11.9 s on a tR2 terminator. 3) The study of a promoter sequence's effect: The effect from sequence of the T5 N25 promoter and T5 N25 antiDSR promoter in transcription was evaluated. The transcription bubble sizes of open complex and initial transcribing complex using the T5 N25 antiDSR promoters are larger than the ones from the T5 N25 promoter. The difference in the two promoter sequences does not have an effect on the transcription bubble size of an elongation complex. And elongation and terminator rewinding kinetics are not affected. On the other hand, abortive initiation and promoter escape are affected by the difference in the promoter sequence. 4) The study of the effect of transcription factor-GreB: The effect of transcription factor-GreB on abortive initiation was evaluated by the single-molecule DNA nanomanipulation technique. GreB does not affect the transcription bubble size during abortive initiation, but does reduce the lifetime of initial transcribing complex.
Note (type = degree)
Ph.D.
Note (type = bibliography)
Includes bibliographical references.
Subject (ID = SUBJ1); (authority = RUETD)
Topic
Computational Biology and Molecular Biophysics
Subject (ID = SUBJ2); (authority = ETD-LCSH)
Topic
Genetic transcription
Subject (ID = SUBJ3); (authority = ETD-LCSH)
Topic
DNA polymerases
RelatedItem (type = host)
TitleInfo
Title
Graduate School - New Brunswick Electronic Theses and Dissertations
Identifier (type = local)
rucore19991600001
Identifier (type = hdl)
http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.16728
Identifier
ETD_509
Location
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NjNbRU
Identifier (type = doi)
doi:10.7282/T3WW7J3R
Genre (authority = ExL-Esploro)
ETD doctoral
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The author owns the copyright to this work.
Copyright
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Open
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Name
Chen-Yu Liu
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Copyright holder
Affiliation
Rutgers University. Graduate School - New Brunswick
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Non-exclusive ETD license
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Author Agreement License
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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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