Mechanisms of Transcription in Microorganisms
Transcription is the central step, and a major
regulatory checkpoint of gene expression. Defective transcription regulation is
the common cause of aberrant growth and development and may result in malignant
transformation. Transcription is carried out by DNA-dependent RNA
polymerasesÑlarge, multisubunit molecular machines. Understanding RNA
polymerase (RNAP) structure and function is a key to understanding gene
expression in molecular detail. The long-term objective of our research is to
uncover the molecular basis of the transcription mechanism and regulation
through structure-functional analysis of RNAP and associated proteins.
Achievement of this goal would be highly significant to the advancement of
basic knowledge about gene expression. As experimental systems we use bacterial
RNAP from Escherichia coli, thermophylic
Thermus aquaticus, plant pathogen
Xanthomonas oryzae, gastric
pathogen Helicobacter pylori, and
RNAP I from yeast.
The following research projects were actively pursued
during the last year:
i. Structure-based
analysis of T. aquaticus RNAPand E.
coli RNAP.
ii. The role of Csp (cold-shock) proteins in
transcription antitermination.
iii. RNAP modifications during bacteriophage development.
iv. Genomics of bacteriophages infecting select agent
bacteria.
The following results were obtained:
i. In collaboration with the group of Dr. Martin Buck
(Imperial College, London, UK) we investigated the effects of several RNAP core
mutations that have large effects on promoter complex formation by s70
RNAP holoenzyme on s54 RNAP holoenzyme promoter complex formation.
The results indicate that s70 RNAP holoenzyme and s54
RNAP holoenzymes utilize similar pathways for active promoter complex formation
despite the lack of evolutionary relation between s70 and s54.
In
collaboration with the group of Raul Salomon, University of Tucuman, Argentina,
we characterized the RNAP binding site for cyclopeptide antibiotic microcin
J25. The results indicate that microcin J25 inhibits transcription through a
novel mechanism, by binding in RNAP secondary channel and obstructing NTP
substrate access to the enzymeÕs catalytic center.
ii. In collaboration with the group of Dr. M. Inouye
(UMDNJ) we discovered that proteins of the ubiquitous Csp (Cold Shock Protein)
family are transcription antiterminators. We further demonstrated that this
novel biochemical activity of Csp proteins is crucial for their physiological
function during cold shock. Mutant Csp proteins that are unable to
antiterminate transcription in vivo
and in vitro were selected. These
mutants were shown to be defective in cold shock response and were unable to
melt secondary RNA structures in vitro. Fine biochemical analysis of melting intermediates trapped by Csp
mutations sheds light on the mechanism of RNA and DNA melting by these
proteins.
iii. We discovered a novel RNAP inhibitor encoded by bacteriophage Xp10,
which infects X. oryzae. We
completed the biochemical analysis of the molecular mechanism of transcription
inhibition by this novel protein. To better understand the physiological role
of this protein, we had determined genomic sequence of Xp10. Xp10 turned out to
be a highly unusual phage; Xp10 had arisen through a recombination event
between a T7-like phage and a lambdoid phage. Currently, we use the methods of
genomics to understand Xp10 gene expression.
We
performed biochemical and biophysical analyses of transcription activation and
transcription inhibition by bacteriophage T4 AsiA protein. The results
uncovered an unexpectedly complex mechanism of AsiA interaction with its target
RNAP subunit, s70. The results also revealed that AsiA changes its
oligomeric state upon interaction with its target.
iv.
We are determining genomic sequences
of several lytic bacteriophages that infect Yersinia pestis, a bacterium that causes plague. We perform
biochemical and bioinformatics analysis of viral sequences to uncover
phage-encoded cytotoxic functions.
Lab Members:
Dr. Konstantin Severinov
Dr. Konstantin Kuznedelov
Dr. Leonid Minakhin
Dr. Elena Severinova
Dr. Tatyana Naryshkina
Dr. Ekaterina Semonova
Dr. Marina Zakharova
Yuliya Yuzenkova
Nicolas Zenkin
Dhruti Savalia