Associate Professor
Member, Center for RNA Biology
Member, MBI
Member, OSBP
The focal point of the research in our lab is RNA polymerase (RNAP), the enzyme that is responsible for the first step in gene expression, mRNA synthesis. RNAP accomplishes this task during the transcription cycle that is composed of three major steps: initiation, elongation, and termination. All these steps are subject to elaborate control by numerous regulatory proteins and small effectors. RNAP is also an attractive target for antibacterial drugs. Using a combination of biochemical, genetic, and structural approaches, we are currently working on several projects:
Substrate selection by RNAP
To faithfully transmit genetic information from genome to proteome form, RNAP must synthesize the nascent RNA with high fidelity. Fidelity mechanisms are well-studied in DNA polymerases, and to a lesser extent in single-subunit (phage T7) RNAPs. However, the mechanism of substrate selection by multi-subunit enzymes remains to be elucidated. We are utilizing structure-based mutagenesis and a combination of in vivo and in vitro analyses to study how the correct nucleotide are selected by RNAP, and have already obtained a set of E. coli RNAP variants with altered substrate-selection properties.
Mechanism and regulation of RNA chain elongation and termination
The rate of transcription is determined by the nucleic acid signals that serve as regulatory checkpoints at which RNAP could be modified by action of auxiliary factors, and therefore determine the gene expression patterns in all organisms. We want to determine how certain DNA and RNA sequences trigger RNAP isomerization into an un-reactive, slow state, which is characterized by a dramatic decrease in the rate of nucleotide addition, and is a likely target for elongation factors (such as NusA, NusG and RfaH). We study how RNAP itself recognizes transcription roadblocks and how auxiliary factors affect its behavior.
RfaH, an elongation enhancer and a virulence factor
Efficient synthesis of long RNA messages relies on transcription factors that allow RNAP to overcome transcription roadblocks. RfaH is a bacterial protein that enables RNAP to transcribe through long operons encoding toxins, antibiotics, capsules, lipopolysaccharide core, and F-pili, all of which are molecules that contribute to pathogenesis. We have obtained the X-ray structure of RfaH, which identifies it as the first example of a bacterial chameleon protein, defined the RfaH binding site on RNAP and demonstrated that RfaH insulates the transcription complex against the sigma-induced pausing elongation. Our long-term goals are to elucidate the molecular mechanism by which RfaH "switches" RNAP into a highly processive state and to test the RfaH role in coupling of transcription and translation.
A cover illustration from Nucleic Acids Research 35(17). Virulence regulator RfaH (shown here in green) suppresses pausing by the bacterial transcription elongation complex composed of RNA polymerase (grey), DNA strands (red and blue), and the nascent RNA transcript (yellow).
Molecular mechanisms of RNAP inhibitors
Inhibitors of bacterial RNAP are used as antibiotics to treat bacterial infections and in research to gain insights into molecular mechanisms that regulate transcription. We are working on the mechanism of RNAP inhibition by streptolydigin, rifamycins, tagetitoxin, CBRs, etc. We perform detailed analysis of the mechanism of action of these inhibitors by a combination of genetic and biochemical techniques.
Regulation of RNA polymerase through secondary channel
RNAP secondary channel postulated to facilitate delivery of substrate NTPs to the active site appears to facilitate access of other small molecules and auxiliary factors to the catalytic center of the enzyme. We study the transcriptional control by the alarmon ppGpp and its protein cofactor DksA, inhibitor of chloroplast development tagetitoxin, RNA cleavage Gre factors, all of which utilize the secondary channel as the only accessible venue connecting the RNAP active site to the surface of the enzyme.
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Belogurov G.A., Sevostyanova A., Svetlov V., Artsimovitch I. (2010) Functional regions of the N-terminal domain of the antiterminator RfaH. Mol. Microbiol. , in press.
Miropolskaya N., Artsimovitch I., Klimašauskas S., Nikiforov V., Kulbachinskiy (2009) Allosteric control of catalysis by the F-loop of RNA polymerase. Proc. Natl. Acad. Sci. USA 106(45):18942-7.
Kulaeva O.I., Gaykalova D.A., Pestov N.A., Golovastov V.V., Vassylyev D.G., Artsimovitch I., Studitsky V.M. (2009) Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II. Nat. Struct. Mol. Biol. 16(12):1272-8.
Belogurov G.A., Mooney R.A., Svetlov V., Landick R., Artsimovitch I. (2009) Functional specialization of transcription elongation factors. EMBO J. 28(2):112-22.
Artsimovitch I., Henkin T. M.. (2009) In vitro approaches to analysis of transcription termination. Methods 47: 37-43.
Belogurov G.A., Vassylyeva M.N. Sevostyanova A., Xiang A., Lira R., Webber S., Klyuyev S., Artsimovitch I., Vassylyev D.G. (2009) Transcription inactivation through local refolding of the RNA polymerase structure. Nature 457(7227): 332-335.
Artsimovitch I. (2008) Post-initiation control by the initiation factor sigma. Mol Microbiol. 68(1):1-3.
Sevostyanova A., Svetlov V., Vassylyev D.G., Artsimovitch I. (2008) The elongation factor RfaH and the initiation factor sigma bind to the same site on the transcription elongation complex. Proc. Natl. Acad. Sci. USA 105(3):865-70.
Artsimovitch I., Vassylyev D.G. (2007) Merging the RNA and DNA worlds. Nat. Struct. Mol. Biol. 14(12):1122-3.
Vassylyeva M.N., Svetlov V., Dearborn A.D., Klyuyev S., Artsimovitch I., Vassylyev D.G. (2007) The carboxy- terminal coiled-coil of the RNA polymerase beta'-subunit is the main binding site for Gre factors. EMBO Rep. 8(11):1038-43.
Svetlov V., Belogurov G.A., Shabrova E., Vassylyev D.G., Artsimovitch I. (2007) Allosteric control of the RNA polymerase by the elongation factor RfaH. Nucleic Acids Res. 35(17):5694-705.
Vassylyev D.G., Vassylyeva M.N., Zhang J., Palangat M., Artsimovitch I., Landick R. (2007). Structural basis for substrate loading in bacterial RNA polymerase. Nature, 448(7150):163-8.
Vassylyev D.G., Vassylyeva M.N., Perederina A., Tahirov T.H., Artsimovitch I. (2007). Structural basis for transcription elongation by bacterial RNA polymerase. Nature, 448(7150):157-62.
Belogurov G.A., Vassylyeva M. N. , Svetlov V., Klyuyev S., Grishin N. V., Vassylyev D.G., and Artsimovitch I. (2007). Structural Basis for Converting a General Transcription Factor into an Operon-Specific Virulence Regulator. Mol Cell, 26: 117-129.
Vassylev D.G., Svetlov. V., Vassylyeva M.N., Perederina A., Igarashi N., Matsugaki N., Wakatsuki S., and Artsimovitch I. (2005). Structural basis for transcription inhibition by tagetitoxin. Nat Struct Mol Biol. Dec;12(12):1086-93.
Artsimovitch I., M. N. Vassylyeva, D. Svetlov, V. Svetlov, A. Perederina, N. Igarashi, N. Matsugaki, S. Wakatsuki, T. H. Tahirov, and D. G. Vassylyev. (2005). Allosteric Modulation of the RNA Polymerase Catalytic Reaction Is an Essential Component of Transcription Control by Rifamycins. Cell, 122:351-363.
Toulme F., Mosrin-Huaman C., Artsimovitch I., and Rahmouni A.R. (2005). Transcriptional pausing in vivo: a nascent RNA hairpin restricts lateral movements of RNA polymerase in both forward and reverse directions. J. Mol. Biol., 351:39-51.
Svetlov, V., Vassylyev, D.G. and Artsimovitch, I (2004). Discrimination against deoxyribonucleotide substrates by bacterial RNA polymerase. J. Biol. Chem. 279:38087-38090.
Artsimovitch, I. (2004). Control of transcription termination and antitermination. In: The Bacterial Chromosome (N. P. Higgins, ed.), American Society for Microbiology, Washington, D.C. pp. 311-326.
Perederina A., Svetlov V., Vassylyeva M.N., Tahirov T.H., Yokoyama S., Artsimovitch I., and Vassylyev D.G. (2004). Regulation through the secondary channel-structural framework for ppGpp-DksA synergism during transcription. Cell, 118:297-309.
Artsimovitch, I.V. Patlan, S. Sekine, M. N. Vassylyeva, T. Hosaka, K. Ochi, S. Yokoyama, D. G. Vassylyev. (2004). Structural basis for transcription regulation by alarmone ppGpp. Cell, 117:299-310.
Carter H.D., Svetlov V., and Artsimovitch I. (2004). Highly divergent RfaH orthologs from pathogenic proteobacteria can substitute for the Escherichia coli RfaH both in vivo and in vitro . J. Bacteriol., 186:2829-2840.
Artsimovitch I., Chu C., Lynch A.S., and Landick R. (2003). A new class of bacterial RNA polymerase inhibitors affects nucleotide addition, Science, 2003, 302:650-654.
Artsimovitch I., and Landick R. (2002). The transcriptional Regulator RfaH Stimulates RNA Chain Elongation after Recruitment to Elongation Complexes by the Exposed Nontemplate DNA Strand. Cell 109:193-203. |
