Transcription is the first step in gene expression and a target for numerous regulatory factors: signals in the transcribed DNA, and in the nascent mRNA, proteins, low molecular weight effectors, etc. One of the most important goals in transcription studies now is identification and "high-resolution" characterization of "hot spots" on RNAP that mediate its interactions with regulators;these sites not only determine the patterns of gene expression, but could also serve as targets for the rational design of antibiotics. Studies of bacterial RNAP complexed with antibiotics should provide short-cuts to the development of effect drug therapies, and a unique opportunity to study dynamic regulatory mechanisms using a "static" crystallographic approach. Among the identified regulatory "hot-spots", the secondary channel (SC) is of special interest. This pore is a major entrance for incoming substrates and accommodates the extruding 39-end of the RNA during transcriptional pausing and arrest. Recently, the SC has emerged as a major binding site for a rapidly expanding group of transcription regulators and catalytic co-factors. This pore apparently can accommodate several accessory proteins: DksA, transcript cleavage Gre-factors, Gfh1, DksA, as well as small RNAP effectors/ antibiotics pyrophosphate, non-complementary NTPs, tagetitoxin, microcin and ppGpp, and thus mediates control of all steps in transcription, from initiation to termination. The goal of this project is to elucidate the basic mechanisms of regulation of transcription elongation through the SC based on high-resolution structural analysis. We will study two model systems, E. coli and T. thermophilus. We propose the following specific aims: 1. We will determine the structures of the EC containing a full transcription bubble in the pre-translocated and backtracked states, and in the presence and absence of the small molecule effectors/inihibitors. 2. We will determine the EC structures with various "SC" transcription factors. The Gre-factors, Gfh1, DksA are among our major targets. Our long-term goal is to elucidate the mechanism of transcription through determining the structures of complexes that represent functional intermediates in the transcription cycle and analysis of the regulatory interactions that ultimately determine the patterns of gene expression in all organisms. We plan to integrate all the available biochemical and biophysical data to build a comprehensive model of the transcription apparatus by elucidating the continuum of structures, from initiation complexes formed at various promoters to those regulatory states that exist during processive elongation. We are particularly interested in elucidation of the structures and mechanisms of the transcription factors (in particular, antibiotics) acting at different steps in transcription. The proposed study represents an essential step in this direction.

Public Health Relevance

Transcription is the first step in gene expression and a target for numerous regulatory factors. Elongation is a most long-lived and highly processive phase of transcription: the elongation complex (EC) is capable of uninterrupted synthesis of thousands nt-long RNA chains. High-resolution structural studies of the bacterial EC appear of central importance for understanding the basic principles of transcription and regulation of gene expression that in turn is essential for design of the new effective anti-bacterial agents. In particular, studies of bacterial RNAP complexed with antibiotics constitute a crucial subset of the structural projects which may provide a short-cut to medicine on one hand and a unique opportunity to study dynamic regulatory mechanisms using a "static" crystallographic approach on the other.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM074840-07
Application #
8292039
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Preusch, Peter C
Project Start
2005-07-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
7
Fiscal Year
2012
Total Cost
$396,689
Indirect Cost
$125,912
Name
University of Alabama Birmingham
Department
Biochemistry
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Klyuyev, Sergiy; Vassylyev, Dmitry G (2012) The binding site and mechanism of the RNA polymerase inhibitor tagetitoxin: an issue open to debate. Transcription 3:46-50
Tsukazaki, Tomoya; Mori, Hiroyuki; Echizen, Yuka et al. (2011) Structure and function of a membrane component SecDF that enhances protein export. Nature 474:235-8
Pupov, Danil; Miropolskaya, Nataliya; Sevostyanova, Anastasiya et al. (2010) Multiple roles of the RNA polymerase {beta}' SW2 region in transcription initiation, promoter escape, and RNA elongation. Nucleic Acids Res 38:5784-96
Zhao, Bin; Lei, Li; Vassylyev, Dmitry G et al. (2009) Crystal structure of albaflavenone monooxygenase containing a moonlighting terpene synthase active site. J Biol Chem 284:36711-9
Vassylyev, Dmitry G (2009) Elongation by RNA polymerase: a race through roadblocks. Curr Opin Struct Biol 19:691-700
Huang, Ying; Ji, Lijuan; Huang, Qichen et al. (2009) Structural insights into mechanisms of the small RNA methyltransferase HEN1. Nature 461:823-7
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
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
Miropolskaya, Nataliya; Artsimovitch, Irina; Klimasauskas, Saulius et al. (2009) Allosteric control of catalysis by the F loop of RNA polymerase. Proc Natl Acad Sci U S A 106:18942-7
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

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