RNA polymerase (RNAP), as the central enzyme of gene expression, is the target of gene regulators. The long-term objectives of this research are to understand how RNAP functions as a molecular machine and t develop methods for intervening in gene expression. We intend to (1) describe in detail how specific amino acid residues and structural features of the active center and regions proximal to the active center function in the catalytic reactions of RNA synthesis and degradation as well as in the regulation of RNA synthesis; (2) describe the mechanism by which RNA polymerase selects nucleotide triphosphate substrates by tracking the route of substrate into the active center; and (3) characterize new RNA polymerase inhibitors previously obtained by screening libraries of small compounds and evaluate those inhibitors as potential tools for investigating of the RNA polymerase catalytic mechanism. Some of these compounds may also serve as lead compounds for antimicrobial development. The reactions of RNA synthesis and degradation will be studied in specifically designed RNA polymerase functional intermediates that are optimal for each particular catalytic activity. The effects of active-center mutations and tested inhibitors on RNA synthesis and degradation will be characterized. Complexes of functional intermediates will be studied using the arsenal of approaches we developed previously, which include chemical nucleic acid-protein crosslinking, Fe2+mediated affinity footprinting, genetically engineered mutations, and discriminative biochemical assays. Moreover, new luminescence-based approaches will be developed to follow dynamic transitions that accompany the nucleotide addition cycle of RNA polymerase. The results will be interpreted in the context of high resolution X-ray structures of RNA polymerase functional complexes using molecular modeling. The data generated by this project are expected to provide new insights into the basic catalytic mechanism of RNA synthesis and its regulation. In addition, the knowledge gained in this study will assist rational design of new inhibitors of RNA polymerase, which is a proven target for antimicrobial chemotherapy.
The reaction mechanism of RNA polymerase will be characterized in detail using a variety of biochemical and biophysical methods that include chemical cross-linking, affinity footprinting, genetic mutations, and new luminescence approaches. This work will be extended by characterizing specific small-molecule inhibitors previously obtained. Some of these inhibitors may serve as lead compounds for developing new RNA polymerase-targeted antimicrobials. ? ? ?
| 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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