The properties of a cell are determined by the information encoded in its genome. Understanding how such information is differentially and dynamically retrieved in response to environmental changes and cellular needs is a major challenge facing molecular biology. The focal point of our research is a multisubunit RNA polymerase (RNAP), the enzyme that carries out the first step in gene expression, synthesis of RNA. By importance, ancienty and conservation among the life kingdoms RNAP rivals the ribosome, yet is considerably less complex and is amenable to kinetics and mechanistic studies in well-defined in vitro systems.

We pursue three interrelated lines of research:

The mechanisms of chemical catalysis and movement. RNAP is one of the nature's most processive enzyme capable of synthesizing RNAs tens of thousands nucleotides long without ever releasing the transcript. RNAP functions as a molecular motor and a helicase; its movement is ultimately powered by the free energy liberated as nucleoside triphosphates are condensed into the nascent RNA and pyrophosphate is released. We perform wide-scale studies of RNAP catalytic mechanism with emphasis on translocation, a step specific to processive polymerases and motor enzymes. The translocation rates are inferred from detailed kinetic studies of single and multiple nucleotide additions. We are also developing assays to monitor translocation directly on the millisecond timescale, with sub-nanometer resolution, and my group will use this technique extensively to study the kinetics, mechanics, and energetics of RNAP translocation.

Transcription elongation factors. Expression of majority of genes is regulated at the stage of transcription. Cellular RNAPs are controlled by a variety of inputs such as nucleic acid signals, numerous protein factors, and small molecules. In collaboration with Dr. Irina Artsimovitch from Ohio State University, we will continue studies of transcription elongation factors. These proteins modulate the rate of RNA synthesis and alter the enzyme's response to regulatory nucleic acid signals, such as pauses or terminators, but their detailed mechanisms are yet unknown. Understanding the means by which transcription factors modulate RNAP catalytic efficiency and translocation kinetics helps us to unravel the basic mechanism of this complex molecular machine.

Evolutionary studies of transcription apparatus. All cellular RNAPs are multisubunit enzymes sharing homologous catalytic cores totaling ~2,500 amino acids. RNAP is arguably the largest protein assembly that is structurally and functionally conserved in all three life kingdoms. In prokaryotes, the core subunits are encoded by single copy genes, whereas in eukaryotic genomes several distinct, deeply rooted, paralogous lineages are present. Considering that the accuracy of molecular phylogenetic inference is critically dependent on the length of the congruent dataset, RNAP is both an excellent phylogenetic marker for inferring species phylogenies and an attractive paradigm for studying the protein evolution. We construct phylogenies from concatenated alignments of RNAP subunits and use them to study the evolution of protein landscape in catalytically important areas of RNAP, as well as the evolution of other transcription-related proteins and the whole organisms.

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 [PubMed]

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 [PubMed]