RNA polymerase (RNAP) is the principal enzyme of gene expression and the target for genetic regulation. The long-term objective of this research is the understanding of the function of bacterial RNAP as a molecular machine at the atomic level of resolution. Specifically, the aims are (1) to build a model of the active center that would assign function to specific amino acid residues, nucleotides and metal ions in reactions of RNA synthesis and degradation and to interpret structurally the interrelationship between these reactions; (2) to characterize conformational transitions in the ternary transcribing complex that modulate catalytic function; (3) to understand transitions in the initial transcribing complex that take place during initial buildup of the nascent transcript, the release of the initiation factor sigma and promoter clearance; and (4) to explore the plasticity of RNAP molecule through generation of aptamers so that multiple conformations of RNAP could be captured for crystallographic studies. To these ends, a series of functionally defined complexes will be generated, in which RNAP will be (a) stalled at a particular stage of the transcription process; or (b) complexed with a defined nucleic acid scaffold, or (c) frozen in a complex with a bound aptamer. The complexes will be studied using chemical nucleic acid-protein crosslinks, genetically engineered mutations in RNAP, and discriminative biochemical assays. The results will be interpreted with the aid of molecular modeling. The understanding of the basic transcription mechanisms generated in this research is a prerequisite for molecular interpretation of regulatory phenomena and the development of interventions into gene expression. In addition, knowledge gained in these studies will be helpful for rational design and screening of new inhibitors of RNAP, which has been a proven target for anti-microbial therapy.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-MBC-2 (01))
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Tompkins, Laurie
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Public Health Research Institute
United States
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Mustaev, Arkady; Malik, Muhammad; Zhao, Xilin et al. (2014) Fluoroquinolone-gyrase-DNA complexes: two modes of drug binding. J Biol Chem 289:12300-12
Pillai, Shyamala; Krasnoperov, Lev; Mustaev, Arkady (2013) Simple no-chromatography procedure for amine-reactive Eu(3+) luminescent chelates optimal for bioconjugation. J Photochem Photobiol A Chem 255:16-23
Kozlov, Maxim; Nudler, Eugeny; Nikiforov, Vadim et al. (2013) Reactive rifampicin derivative able to damage transcription complex. Bioconjug Chem 24:443-7
Pratt, Ayiasha; Garcia-Effron, Guillermo; Zhao, Yanan et al. (2013) Evaluation of fungal-specific fluorescent labeled echinocandin probes as diagnostic adjuncts. Med Mycol 51:103-7
Kurepina, N; Kreiswirth, B N; Mustaev, A (2013) Growth-inhibitory activity of natural and synthetic isothiocyanates against representative human microbial pathogens. J Appl Microbiol 115:943-54
Wirpsza, Laura; Krasnoperov, Lev; Mustaev, Arkady (2013) New quinolone-based thiol-reactive lanthanide luminescent probes. J Photochem Photobiol A Chem 251:30-37
Sosunova, Ekaterina; Sosunov, Vasily; Epshtein, Vitaly et al. (2013) Control of transcriptional fidelity by active center tuning as derived from RNA polymerase endonuclease reaction. J Biol Chem 288:6688-703
Pillai, Shyamala; Kozlov, Maxim; Marras, Salvatore A E et al. (2012) New cross-linking quinoline and quinolone derivatives for sensitive fluorescent labeling. J Fluoresc 22:1021-32
Wirpsza, Laura; Pillai, Shyamala; Batish, Mona et al. (2012) Highly bright avidin-based affinity probes carrying multiple lanthanide chelates. J Photochem Photobiol B 116:22-9
Malik, Muhammad; Marks, Kevin R; Mustaev, Arkady et al. (2011) Fluoroquinolone and quinazolinedione activities against wild-type and gyrase mutant strains of Mycobacterium smegmatis. Antimicrob Agents Chemother 55:2335-43

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