RNA polymerase (RNAP) is the central enzyme of transcription and a major target of regulation. Each stage of the transcription cycle can be regulated through interactions of RNAP with various transcription factors. Bacteriophages (phages) evolved efficient regulatory mechanisms to convert host transcription machinery to serve the needs of the phage. Since phages are the most abundant and diverse life form in the Biosphere, the gamut of phage-encoded transcription factors is practically inexhaustible. Studies of such phage-encoded proteins provided and continue to provide paradigms of genetic regulation of general biological significance. Studies of phages infecting thermophilic bacteria, organisms whose proteins form superior crystals, have an added advantage, for they make it possible to obtain structural information about phage-encoded regulators in complex with their cellular target, RNAP. Our goal is to study bacteriophage-dependent regulation of transcription in thermophilic Thermus eubacteria whose RNAPs have been crystallized. Despite recent advances in phage genomics, only a few thermophilic phages have been completely sequenced. Recently, the genomes of 3 phages infecting T. thermophilus (Tth) were determined in our laboratory. The process of Tth infection by each of these phages will be studied to achieve the following specific aims. 1. Phage-encoded proteins that interact with Tth RNAP will be identified by mass- spectrometric analysis of proteins that co-purify with affinity-tagged host RNAP during a highly-efficient immunoaffinity purification. 2. Identified phage proteins will be functionally characterized in a fully recombinant Tth RNAP in vitro transcription system that we developed; the molecular mechanisms of action of the of phage regulators and their binding sites on host RNAP will be determined. To deeper understand the mechanism of transcription regulation we will perform structural analysis of complexes between phage regulators and host RNAP. The structural work will be carried out as a collaboration with two leading crystallographic groups. As a result of proposed collaborative studies, novel phage-encoded RNAP-binding transcription proteins will be identified and characterized functionally and structurally. The results will uncover novel transcription regulation mechanisms and will reveal RNAP sites that can be used as potential targets for development of drugs that act by affecting bacterial RNAP, a validated drug target. Bacteriophages evolved a remarkably diverse array of regulatory proteins that bind to and inhibit bacterial RNA polymerase (RNAP). Bacterial RNAP is a central enzyme in gene expression and is a validated antibacterial drug target: Rifampicin, an RNAP inhibitor, currently is a frontline drug against tuberculosis. Thus, identification and characterization of phage proteins that bind bacterial RNAP and inhibit gene expression will have a tremendous potential for identifying new antibiotic proteins and for rational and effective antibiotic design. ? ? ?

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Beanan, Maureen J
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Rutgers University
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New Brunswick
United States
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Berdygulova, Zhanna; Westblade, Lars F; Florens, Laurence et al. (2011) Temporal regulation of gene expression of the Thermus thermophilus bacteriophage P23-45. J Mol Biol 405:125-42
Minakhin, Leonid; Goel, Manisha; Berdygulova, Zhanna et al. (2008) Genome comparison and proteomic characterization of Thermus thermophilus bacteriophages P23-45 and P74-26: siphoviruses with triplex-forming sequences and the longest known tails. J Mol Biol 378:468-80