Our long-term goal is to understand the function and regulation of bacterial DNA-dependent RNA polymerase (RNAP) in molecular detail. Bacterial viruses--phages--evolved elaborate mechanisms to regulate host transcription in order to make it serve the needs of the virus. The variety of phages and the number of regulatory mechanisms that they evolved vastly exceeds the variety of bacterial regulatory mechanisms. Phage regulatory systems are compact, robust, and efficient (i.e., phage-encoded proteins are small, they interact with host RNAP tightly, and their regulatory effects are strong). Studies of only a handful of modifications of host RNAP by proteins encoded by phages infecting well-studied bacteria such as E. coli provided paradigmatic examples of regulation of gene expression that are applicable to both bacteria and higher organisms. However, the structural understanding of regulation of RNAP function by phage proteins is generally lacking. The goal of this research is i) to identify proteins encoded by thermophages (phages infecting bacteria of the Thermus genus) that bind to host RNAP;ii) to determine the binding sites of these proteins and functional consequences of their binding, and iii) in collaboration with leading structural groups, to determine the structures of complexes between phage proteins and Thermus RNAP, the only bacterial RNAP that forms diffracting crystals and for which high-resolution structural information is available. The proposed strategy allows, for the first time, to directly relate the function of RNAP-binding transcription factors and the structure of their complexes with the target enzyme. Detailed characterization of new phage-encoded transcription regulators that interact with different subunits of RNAP and affect different stages of the transcription cycle will provide novel molecular probes to better understand RNAP mechanism and regulation and to uncover RNAP sites that can be targets for drug design. Whenever possible, the role of thermophage proteins that bind host RNAP in viral development will be determined.
During infection by bacterial viruses (phages) the gene transcription enzyme of bacterial host -- RNA polymerase (RNAP) -- stops expressing host genes and starts expressing viral genes;this change is often caused by the binding of phage proteins to host RNAP. We propose to identify and characterize, both functionally and structurally, several phage proteins that bind to and change the activity of RNAP from Thermus bacteria, the only bacterial RNAP which can be crystallized and for which the structure is known. The results will allow, for the first time, to directly relate the function and structure of transcription regulators, lead to better understanding of bacterial transcription and help design new compounds that inhibit bacterial RNAP, a validated target of antibiotics.
|Mekler, Vladimir; Minakhin, Leonid; Borukhov, Sergei et al. (2014) Coupling of downstream RNA polymerase-promoter interactions with formation of catalytically competent transcription initiation complex. J Mol Biol 426:3973-84|
|Liu, Bing; Shadrin, Andrey; Sheppard, Carol et al. (2014) A bacteriophage transcription regulator inhibits bacterial transcription initiation by ?-factor displacement. Nucleic Acids Res 42:4294-305|
|Zorov, Savva; Yuzenkova, Yulia; Nikiforov, Vadim et al. (2014) Antibiotic streptolydigin requires noncatalytic Mg2+ for binding to RNA polymerase. Antimicrob Agents Chemother 58:1420-4|
|Klimuk, Evgeny; Akulenko, Natalia; Makarova, Kira S et al. (2013) Host RNA polymerase inhibitors encoded by ?KMV-like phages of Pseudomonas. Virology 436:67-74|
|Sheppard, Carol; James, Ellen; Barton, Geraint et al. (2013) A non-bacterial transcription factor inhibits bacterial transcription by a multipronged mechanism. RNA Biol 10:495-501|
|Mekler, Vladimir; Severinov, Konstantin (2013) Cooperativity and interaction energy threshold effects in recognition of the -10 promoter element by bacterial RNA polymerase. Nucleic Acids Res 41:7276-85|
|Westra, Edze R; van Erp, Paul B G; Kunne, Tim et al. (2012) CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol Cell 46:595-605|
|Pavlova, Olga; Lavysh, Daria; Klimuk, Evgeny et al. (2012) Temporal regulation of gene expression of the Escherichia coli bacteriophage phiEco32. J Mol Biol 416:389-99|
|Mekler, Vladimir; Minakhin, Leonid; Sheppard, Carol et al. (2011) Molecular mechanism of transcription inhibition by phage T7 gp2 protein. J Mol Biol 413:1016-27|
|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|
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