Date Approved
10-2013
Document Type
Dissertation
Degree Name
PhD in Cell and Molecular Biology
Department
Cell Biology
College
Graduate School of Biomedical Sciences
Funder
UMDNJ/Rowan Research Foundation Grants and the National Institutes of Health
First Advisor
Sergei Borukhov, PhD
Committee Member 1
Randy Strich, PhD
Committee Member 2
William McAllister, Ph
Committee Member 3
Dimitri Pestov, PhD
Committee Member 4
Venkataswar Venkataraman, PhD
Subject(s)
RNA Polymerases; RNA Cleavage; Bacteria; Transcription Factors
Disciplines
Bacteriology | Cell Biology | Laboratory and Basic Science Research | Molecular Biology | Molecular Genetics
Abstract
The activity of RNA polymerase (RNAP), the key enzyme of transcription process, is regulated by a large number of transcription factors acting at each step of the transcription cycle. Our research project is focused on studying the structure and function of bacterial transcript cleavage factors GreA, GreB and the stringent response regulator DksA. Gre factors bind in the secondary channel of RNAP in backtracked (inactive) ternary elongation complexes and stimulate the enzyme's intrinsic RNase activity, which is required for suppression of transcription pause and arrest, enhancement of transcription fidelity, and efficient promoter escape. DksA also acts through the RNAP secondary channel and it functions as a critical cofactor for ppGpp-mediated positive and negative regulation of transcription. DksA does not stimulate transcript cleavage; instead, depending on the promoter, it can destabilize the RNAP-DNA open promoter complexes. To better understand the molecular mechanism of action of these factors, we characterized the molecular interfaces of GreA-RNAP and DksA-RNAP complexes using site-directed mutagenesis and site-specific protein-protein crosslinking. We determined that Gre factors use the tip of the RNAP f3' coiled-coil element (f3' RH) as a major docking site; upon binding to RNAP, the activity of Gre factors is regulated by the flexible domains of the secondary channel (Trigger Loop, TL and SI3). We showed that, unlike Gre factors, DksA interacts cooperatively with two structural elements of the RNAP, f3' RH and f3 SI I. We also identified two key residues in the f3 subunit of RNAP that are essential for DksA activity but not for binding. In addition, we determined the role of different conformations of the RNAP TL in major transcriptional activities. We showed that the “closed” conformation of TL is necessary and sufficient for NTP binding, catalysis and translocation, however it restricts NTP diffusion to the active center of RNAP and prevents binding of transcript cleavage factors. Remarkably, "closing" of the TL dramatically increases fidelity of transcription. The "'open" conformation of TL is incompatible with RNAP polymerization activity. However, it is required for binding and functioning of transcript cleavage factors. Reversible folding of TL from the “closed” to the '"open" state is essential for maximal rates of processive elongation.
Recommended Citation
Parshin, Andrey, "Bacterial Transcription Factors GreA, GreB and DksA: Characterization of Their Interaction with RNA Polymerase and Molecular Mechanism of Action" (2013). Graduate School of Biomedical Sciences Theses and Dissertations. 67.
https://rdw.rowan.edu/gsbs_etd/67
Included in
Bacteriology Commons, Cell Biology Commons, Laboratory and Basic Science Research Commons, Molecular Biology Commons, Molecular Genetics Commons
