Belogurov G.A., Artsimovitch I. (2015) Regulation of transcript elongation. Annu. Rev. Microbiol. 69: 49-69.
RNA Polymerase Group: Molecular Mechanisms of Transcription
University of Turku, Department of Biochemistry, Tykistökatu 6, 20520 Turku, Finland.
Multisubunit RNA polymerase
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 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. A recent explosion in high-resolution (2.0-3.5 Å) structures of RNAP and transcription complexes puts us in an excellent position to begin addressing the key mechanistic questions in the field of transcription. We study RNAP catalytic mechanism with emphasis on translocation, a step specific to processive polymerases and motor enzymes.
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. Transcription elongation factors 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 employ molecular phylogenetic analyses 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.
Our laboratory has established methods for expression, purification and analysis of the multisubunit RNAPs from Bacteria (Escherichia coli, Thermus thermophilus, Fibrobacter succinogenes and the list is growing) and Eukaryotes (Saccharomyces cerevisiae RNA polymerase II). As a part of our studies of transcription inhibitors we further expanded our experimental portfolio to include the single-subunit RNAPs from mitochondria (Homo sapiens and Saccharomyces cerevisiae) and viruses (3Dpol from poliovirus, CVB3 from coxsackievirus).
Belogurov G.A., Artsimovitch I. (2019) The mechanisms of substrate selection, catalysis and translocation by the elongating RNA polymerase. J. Mol. Biol. pii: S0022-2836(19)30326-2. [PubMed]
Turtola M., Mäkinen J.J., Belogurov G.A. (2018) Active site closure stabilizes the backtracked state of RNA polymerase. Nucleic Acids Res. 46(20):10870-10887. [PubMed]
Supplementary Figure S6 in 3D (WebGL in browser):  Option 1   Option 2
Sanz-Murillo M., Xu J., Belogurov G.A., Calvo O., Gil-Carton D., Moreno-Morcillo M., Wang D., Fernández-Tornero C. (2018) Structural basis of RNA polymerase I stalling at UV light-induced DNA damage. Proc. Natl. Acad. Sci. USA 115(36):8972-8977. [PubMed]
Nedialkov Y., Svetlov D., Belogurov G.A., Artsimovitch I. (2018) Locking the non-template DNA to control transcription. Mol. Microbiol. 109(4):445-457. [PubMed]
Figure 4 in 3D (WebGL in browser):   Model 1     Model 2     Model 3     Model 4
Artsimovitch I., Belogurov G.A. (2018) Uneven braking spins RNA polymerase into a pause. Mol. Cell 69(5):723-725. [PubMed]
Animated Figure 1 (GIF)
NandyMazumdar M., Nedialkov Y., Svetlov D., Sevostyanova A., Belogurov G.A., Artsimovitch I. (2016) RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes. Proc. Natl. Acad. Sci. USA 113(52):14994-14999. [PubMed]
Figure 1 in 3D (WebGL in browser):   panel A     panel B
Turtola M., Belogurov G.A., (2016) NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble. eLife 5. pii: e18096. [PubMed]
Figure 8 in 3D (WebGL in browser):   Panel C
Maddalena L.L., Niederholtmeyer H., Turtola M., Swank Z.N., Belogurov G.A., Maerkl S.J. (2016) GreA and GreB enhance expression of Escherichia coli RNA polymerase promoters in a reconstituted transcription-translation system. ACS Synth. Biol. 5(9):929-35. [PubMed]
Esyunina D., Turtola M., Pupov D., Bass I., Klimaðauskas S., Belogurov G., Kulbachinskiy A. (2016) Lineage-specific variations in the trigger loop modulate RNA proofreading by bacterial RNA polymerases. Nucleic Acids Res. 44(3):1298-308. [PubMed]
Belogurov G.A., Artsimovitch I. (2015) Regulation of transcript elongation. Annu. Rev. Microbiol. 69: 49-69. [PubMed]
Artsimovitch I., Belogurov G.A. (2015) Creative math of RNA polymerase III termination: sense plus antisense makes more sense. Mol. Cell 58(6):974-6. [PubMed]
Malinen A.M., Turtola M., Belogurov G.A. (2015) Monitoring translocation of multisubunit RNA polymerase along the DNA with fluorescent base analogues. Methods Mol. Biol. 1276:31-51. [PubMed]
Malinen A.M., NandyMazumdar M., Turtola M., Malmi H., Grocholski T., Artsimovitch I., Belogurov G.A. (2014) CBR antimicrobials alter coupling between the bridge helix and the ß subunit in RNA polymerase. Nat. Commun. 5:3408 doi: 10.1038/ncomms4408. [PubMed]
Malinen A.M., Turtola M., Parthiban M., Vainonen L., Johnson M.S., Belogurov G.A. (2012) Active site opening and closure control translocation of multisubunit RNA polymerase. Nucleic Acids Res. 40(15): 7442-51. [PubMed]