RNA polymerase is the principal enzyme of gene expression and the target for genetic regulation. Its basic structure-functional features are highly conserved among all living organisms. The broad goal of this project is the understanding of the molecular mechanism of RNA polymerase basic function using site directed mutagenesis of evolutionarily invariant amino acids in individual subunits of RNA polymerase from Escherichia coli. For this purpose, single amino acid substitution mutations, linker insertions and in-frame deletions will be generated in the cloned gene rpoB which encodes the core RNA polymerase beta subunit. The mutant RNA polymerase holoenzyme (alpha-2-beta-beta-'sigma) will then be prepared for biochemical characterization by in vitro reconstitution from the individual subunits. The subunits will be obtained by overexpressing them from plasmids carrying the three rpo genes under the control of inducible promoter. The malfunctioning of RNA polymerase caused by the amino acid substitutions will be characterized using an arsenal of in vitro assays which would allow to monitor and quantitate individual steps of the enzyme's functional cycle. At the second stage of this project, compensatory suppressor mutations will be isolated and mapped. This approach is anticipated to provide information about molecular details of promoter binding, the functioning of the enzyme's catalytic center, the nature of processivity, the mechanism of pausing and termination, as well as to, uncover structural elements of the enzyme which carry out these functions.
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| 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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