RNA polymerase is the central enzyme of gene expression in all domains of life. Changes in gene expression patterns punctuate the progress of normal and neoplastic development of eukaryotes and onset of virulence in bacteria. Elongation is the longest and a highly regulated part of transcription cycle. Elongation factors such as PTEF-b and RfaH are implicated in development of cancer in humans and virulence in bacteria, respectively. This project aims at elucidation of the detailed molecular mechanism of transcript elongation by bacterial RNA polymerase by combining the high-resolution structural analysis of elongation intermediates with the state-of-the-art biochemical characterization of the elongation mechanism. Different states of the transcription complex that have been detected by biochemical and single-molecule experiments likely serve as targets for protein factors, small RNAs, nucleotide analogs, antibiotics, and other regulators of gene expression. Structures of RNA polymerase complexes that represent functional intermediates in the transcription cycle will be determined for the two model bacterial systems, Escherichia coli and Thermus thermophilus. The former is the major source of the biochemical and genetic data, whereas the latter afforded the high-resolution RNAP structures.
Specific aims of this study will be as follows. First, to determine organization and structure of the transcription elongation complex at the atomic level. Nucleic acid scaffolds will be used to assemble, characterize, and crystallize functional elongation complexes captured in different conformational states and to solve their structures. Structure-based site-directed mutagenesis will be then applied to examine functional importance of the key elements of the transcription elongation complex. Second, to elucidate the mechanism of substrate selection and catalysis by bacterial RNA polymerase, structures of the transcription elongation complexes with the incoming substrates will be determined by X-ray analysis. Subsequent functional tests will be designed to identify the determinants for nucleotide selection and incorporation; in parallel, genetic approaches will be used to study the regulation of transcriptional fidelity in vivo. Third, to elucidate the elongation complex determinants that control both its high thermodynamic stability and the facile translocation upon nucleotide addition, the effects of systematic variation of the scaffold components and substitutions of the key RNA polymerase residues that are highlighted by the structure will be analyzed

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM074252-03
Application #
7204114
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Tompkins, Laurie
Project Start
2005-04-01
Project End
2010-03-31
Budget Start
2007-04-01
Budget End
2008-03-31
Support Year
3
Fiscal Year
2007
Total Cost
$453,895
Indirect Cost
Name
University of Alabama Birmingham
Department
Biochemistry
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Ha, Kook Sun; Toulokhonov, Innokenti; Vassylyev, Dmitry G et al. (2010) The NusA N-terminal domain is necessary and sufficient for enhancement of transcriptional pausing via interaction with the RNA exit channel of RNA polymerase. J Mol Biol 401:708-25
Belogurov, Georgiy A; Sevostyanova, Anastasia; Svetlov, Vladimir et al. (2010) Functional regions of the N-terminal domain of the antiterminator RfaH. Mol Microbiol 76:286-301
Kulaeva, Olga I; Gaykalova, Daria A; Pestov, Nikolai A et al. (2009) Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II. Nat Struct Mol Biol 16:1272-8
Huang, Ying; Ji, Lijuan; Huang, Qichen et al. (2009) Structural insights into mechanisms of the small RNA methyltransferase HEN1. Nature 461:823-7
Belogurov, Georgiy A; Vassylyeva, Marina N; Sevostyanova, Anastasiya et al. (2009) Transcription inactivation through local refolding of the RNA polymerase structure. Nature 457:332-5
Vassylyev, Dmitry G (2009) Elongation by RNA polymerase: a race through roadblocks. Curr Opin Struct Biol 19:691-700
Kent, Tatyana; Kashkina, Ekaterina; Anikin, Michael et al. (2009) Maintenance of RNA-DNA hybrid length in bacterial RNA polymerases. J Biol Chem 284:13497-504
Sydow, Jasmin F; Brueckner, Florian; Cheung, Alan C M et al. (2009) Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA. Mol Cell 34:710-21
Tsukazaki, Tomoya; Mori, Hiroyuki; Fukai, Shuya et al. (2008) Conformational transition of Sec machinery inferred from bacterial SecYE structures. Nature 455:988-91
Baranovskiy, Andrey G; Babayeva, Nigar D; Liston, Victoria G et al. (2008) X-ray structure of the complex of regulatory subunits of human DNA polymerase delta. Cell Cycle 7:3026-36

Showing the most recent 10 out of 25 publications