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Probing The Dynamics of RNA Polymera...

Bettridge, Kelsey Elaine.

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Probing The Dynamics of RNA Polymerase and Hu in Live E. coli Cells.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Probing The Dynamics of RNA Polymerase and Hu in Live E. coli Cells./
Author:	Bettridge, Kelsey Elaine.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2019,
Description:	185 p.
Notes:	Source: Dissertations Abstracts International, Volume: 81-08, Section: B.
Contained By:	Dissertations Abstracts International81-08B.
Subject:	Microbiology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=27726326
ISBN:	9781392878248

Probing The Dynamics of RNA Polymerase and Hu in Live E. coli Cells.
Bettridge, Kelsey Elaine.

Probing The Dynamics of RNA Polymerase and Hu in Live E. coli Cells. - Ann Arbor : ProQuest Dissertations & Theses, 2019 - 185 p.

Source: Dissertations Abstracts International, Volume: 81-08, Section: B.

Thesis (Ph.D.)--The Johns Hopkins University, 2019.

This item must not be sold to any third party vendors.

Diffusion is at the heart of every biochemical process. Millions of proteins must navigate the heterogeneous, crowded cellular milieu to perform their various tasks. This molecular crowding has a significant effect on the diffusive behavior and kinetic rates of proteins and biochemical reactions. A powerful technique to understand biochemical processes within the context of this heterogeneous environment is single particle tracking (SPT). In Chapter 2, I use SPT to elucidate the dynamics of the RNAP search process and transcription cycle in live E. coli cells. Using FRAP, I find that transcription follows a simple initiation-elongation-termination cycle with kinetic rates that closely match those in the literature. Using SPT, I probed the search process of RNAP and found three diffusive states corresponding to DNA-bound, diffusion within the dense nucleoid, and diffusion within the cytoplasm. RNAP exhibited confinement in each state and displayed a preference for a DNA-bound state, suggesting a grid search strategy. Additionally, RNAP displayed kinetics that were not consistent with steady state kinetics. In Chapter 3, I use SPT to probe the molecular mechanism of HU-mediated chromosome organization. Using genetic mutations that abolish the various binding modes of HU, I find that HUαα and HUαβ displayed differential dynamics. Additionally, HUαα seems primarily responsible for non-specific binding while HUαβ seems primarily responsible for repressor loop formation. The kinetics of HU were highly transient, indicative of their non-specific binding across the nucleoid, and suggested a mechanism by which cumulative forces of thousands of HU are able to achieve chromosomal organization, a marked departure from the long-lived binding of other DNA organization proteins such as histones.

ISBN: 9781392878248Subjects--Topical Terms:

536250
Microbiology.
Subjects--Index Terms:

Biophysics

Probing The Dynamics of RNA Polymerase and Hu in Live E. coli Cells.
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245 1 0 $a Probing The Dynamics of RNA Polymerase and Hu in Live E. coli Cells.
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300 $a 185 p.
500 $a Source: Dissertations Abstracts International, Volume: 81-08, Section: B.
500 $a Advisor: Wolberger, Cynthia.
502 $a Thesis (Ph.D.)--The Johns Hopkins University, 2019.
506 $a This item must not be sold to any third party vendors.
520 $a Diffusion is at the heart of every biochemical process. Millions of proteins must navigate the heterogeneous, crowded cellular milieu to perform their various tasks. This molecular crowding has a significant effect on the diffusive behavior and kinetic rates of proteins and biochemical reactions. A powerful technique to understand biochemical processes within the context of this heterogeneous environment is single particle tracking (SPT). In Chapter 2, I use SPT to elucidate the dynamics of the RNAP search process and transcription cycle in live E. coli cells. Using FRAP, I find that transcription follows a simple initiation-elongation-termination cycle with kinetic rates that closely match those in the literature. Using SPT, I probed the search process of RNAP and found three diffusive states corresponding to DNA-bound, diffusion within the dense nucleoid, and diffusion within the cytoplasm. RNAP exhibited confinement in each state and displayed a preference for a DNA-bound state, suggesting a grid search strategy. Additionally, RNAP displayed kinetics that were not consistent with steady state kinetics. In Chapter 3, I use SPT to probe the molecular mechanism of HU-mediated chromosome organization. Using genetic mutations that abolish the various binding modes of HU, I find that HUαα and HUαβ displayed differential dynamics. Additionally, HUαα seems primarily responsible for non-specific binding while HUαβ seems primarily responsible for repressor loop formation. The kinetics of HU were highly transient, indicative of their non-specific binding across the nucleoid, and suggested a mechanism by which cumulative forces of thousands of HU are able to achieve chromosomal organization, a marked departure from the long-lived binding of other DNA organization proteins such as histones.
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650 4 $a Optics. $3 517925
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653 $a Biophysics
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653 $a Fluorescence microscopy
653 $a Gene regulation
653 $a Chromosome organization
653 $a Nucleoid organization
653 $a RNA polymerase
653 $a DNA-binding protein
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