Kseniya Obraztsova

Date Approved

8-2016

Document Type

Dissertation

Degree Name

PhD in Cell and Molecular Biology

Department

Cell Biology

College

Graduate School of Biomedical Sciences

First Advisor

Sergei Borukhov, PhD

Committee Member 1

Natalia Shcherbik, PhD

Committee Member 2

Mikhail Anikin, PhD

Committee Member 3

Randy Strich, PhD

Committee Member 4

Ronald Ellis, PhD

Subject(s)

Escherichia coli, Osmoregulation, Osmotic Stress Response, Transcriptional Regulatory Element, RNA Polymerases, Transcriptases

Disciplines

Cell Biology | Medical Cell Biology | Medical Molecular Biology | Medicine and Health Sciences | Molecular Biology

Abstract

Escherichia coli adapts to changes in the osmotic environment through the process of osmotic response which entails synthesis of specific enzymes and transporters to accumulate essential osmoprotectants. This process is highly regulated at the level of transcription. Upon cell exposure to high salt, RNA polymerase (RNAP} rapidly dissociates from genomic DNA, while the nucleoid becomes hyper condensed. During the subsequent osmoadaptation phase RNAP reassociates with the DNA and resumes transcription. The mechanisms of transcription initiation, promoter escape and its regulation during osmoadaptation are poorly understood. Here we demonstrate that a highly conserved bacterial regulator, transcript cleavage factor GreA, is essential for cell growth under hyperosmotic conditions. Using chromatin immunoprecipitation (ChlP) approach, we demonstrated that during the osmoadaptation phase, RNAP in cells lacking Gre factors (E. coli BA) undergoes a genome-wide redistribution and accumulates disproportionately at promoters and promoter-proximal regions. At the same time, RNAP binding signal in the downstream coding regions of many genes, including genes of essential functions and genes involved in osmotic response, dramatically decreases. GreA and its homolog GreB relieve genome-wide promoter-proximal stalling/pausing, and restore normal genomic distribution, i.e. gene occupancy, of RNAP. Based on our results, we propose that under hyperosmotic stress RNAP becomes trapped at many promoters and that Gre factors rescue the inactive stalled/paused promoter-proximal complexes, restoring transcriptional activity of RNAP.

In support of this role of Gre, we identified a suppressor mutation in RNAP 13' subunit (rpoC-PSlL) which allows BA- cells to grow under hyperosmotic stress. We showed that, like Gre factors, this mutation decreased the amount of abortive products synthesized on specific promoters in vitro, and suppressed genome wide promoter-proximal stalling induced by osmotic stress in vivo, restoring RNAP occupancy on genome.

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