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Bacterial transcription factors GreA, GreB and DksA: characterization of their interaction with RNA polymerase and molecular mechanism of action

Andrey Parshin
B.Sc., 2004
Samara State University, Russia

Thesis Advisor: Sergei Borukhov, Ph.D.

Cell and Molecular Biology Program

Science Center, Room 290

Thursday, April 18, 2013
12 pm


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-function of bacterial transcript cleavage factors GreA, GreB and 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 B’ coiled-coil element (B’ 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 the DksA interacts cooperatively with the two structural elements of the RNAP, B’ RH and B SI1. We also identified two key residues in B subunit of RNAP that are essential for DksA activity but not for binding. In addition, we determined the rolfde of the 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 “closed” to “open” state is essential for maximal rate of processive elongation.

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