After infection of E. coli by bacteriophage T4, the host RNA polymerase acquires several small phage-induced polypeptides and its alfa subunits are ADP-ribosylated. The role of these modifications in transcription control will be studied using a combination of biochemical, genetic and physiological approaches. We have purified four of the associated polypeptides (15K, 19K, 25K and 29K proteins) as well as RNA polymerases differing in the state of ADP-ribosylation and propose to study their interactions in direct binding assays. The kinetics of in vivo formation of these proteins and their interactions with other intracellular components will be analysed in order to understand the coordination between transcription and other events of T4 development such as DNA replication. We will identify T4 genes involved in the modifications and use their mutants to determine their role in phage development. In vitro experiments are proposed to study the interaction of normal and modified RNA polymerase with individual promoters with emphasis given to the change of specificity of promoter recognition from early to late sites. We have already shown that 25K protein induces late promoter specificity while 15K protein decreases the utilization of early promoters. To understand the molecular basis of the specificity change we will determine the kinetic parameters of promoter functioning with different forms of RNA polymerase. Experiments directed at biochemical identification of T4-induced antitermination mechanism are also proposed. The in vitro experiments will be backed up by the analysis of in vivo functioning of the same transcription sites using recently sequenced tRNA gene region of T4 as the experimental system.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Columbia University (N.Y.)
Schools of Medicine
New York
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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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