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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 (in collaboration
with Dr. Vassylyev's Lab at UAB)
approaches, we are currently working on several projects:
I. 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.
II. 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. We have recently identified the rate-limiting step in the
transcription cycle, a dramatic refolding of the beta' subunit trigger loop into
two trigger helices.
III. RfaH, an elongation enhancer and a virulence factor
Efficient synthesis of long messages relies on transcription factors that allow
RNAP to overcome transcription roadblocks. RfaH is a bacterial antitermination
factor that enables RNAP to transcribe through long polycistronic operons
encoding toxins, antibiotics, capsules, lipopolysaccharide core, and F-pili, all
of which are molecules that contribute to bacterial pathogenesis. We have
obtained the X-ray structure of RfaH, which identifies it as the first example
of a bacterial chameleon protein. We have defined the binding site of RfaH on
RNAP and demonstrated that RfaH insulates the transcription complex against the
sigma-induced pausing elongation. Currently, we are characterizing the RfaH regulon
in E. coli, conducting the comparative analysis of RfaH orthologs from different
bacteria (Y. enterocolitica, V. cholerae, K. pneumoniae, etc.), studying the
molecular mechanism by which RfaH "switches" RNAP into a highly processive
state, and testing if RfaH is involved 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). For further details, including the effects of amino acid
substitutions in RNA polymerase (shown as colored spheres) on RfaH function, see
the article by Svetlov et al. (Nucleic Acids Res. (2007) 35(17), 5694-
5705).
IV. 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 and collaborate with Dr. Vassylyev's Laboratory at UAB to
obtain high-resolution X-ray structures of the Thermus thermophilus RNAP in
complex with different drugs. We plan to use the collected data for design of
novel antibiotics.
V. 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. Alarmon ppGpp,
inhibitor of chloroplast development tagetitoxin, cleavage Gre factors, GfhI,
and their structural analog DksA all have been recently added to the growing
list of transcriptional regulators utilizing secondary channel as the only
accessible venue connecting the RNAP active site to the surface of the enzyme.
Presently we collaborate with Dmitry Vassylyev in elucidating the mechanisms by
which these and other factors regulate activity of RNAP.
People in the lab:
Irina Artsimovitch,
Georgy Belogurov,
Vasil Khomenkov,
Anastasia Sevostiyanova,
Ran Furman
Former Lab Members:
Vladimir Svetlov,
Dmitri Svetlov,
Heather D. Carter,
Elena Shabrova,
Ryan Powell,
Daniele Vicari,
Amy Stockert,
Adrian Lange
Useful stuff:
Expression vector for E. coli RNA polymerase
pIA plasmids list
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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.
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