Research Summary Mechanisms of Transcription in Microorganisms Transcription is the central step, and a major regulatory checkpoint of gene expression. Defective transcription regulation is the common cause of aberrant growth and development and may result in malignant transformation. Transcription is carried out by DNA-dependent RNA polymerases–large, multisubunit molecular machines. Understanding RNA polymerase (RNAP) structure and function is a key to understanding gene expression in molecular detail. The long-term objective of our research is to uncover the molecular basis of the transcription mechanism and regulation through structure-functional analysis of RNAP and associated proteins. In addition, we use bacteriophage development as a model system to study temporal regulation of gene expression and to uncover novel mechanisms of transcription regulation. As experimental systems we use bacterial RNAP from Escherichia coli, thermophilic Thermus aquaticus and T. thermophilus, opportunistic human pathogen Pseudomonas aeruginosa, and plant pathogen Xanthomonas oryzae. The following research projects were actively pursued during the last year: Studies of bacteriophage development We analyzed the process of E. coli RNA polymerase-catalyzed synthesis of replication primer from single-stranded origin of replication of bacteriophage M13. The results define a novel conformation of transcription elongation complex that is formed due to formation of overextended RNA-DNA hybrid during transcription of single-stranded DNA. Similar conformations may arise during transcription of double-stranded cellular DNA and may affect the rate of elongation and be subject to regulation. We completed genomic sequencing and determined the temporal transcription pattern of B. anthracis typing phage Fah. The results showed that the late genes of the phage are expressed with the help of phage-encoded RNA polymerase ? factor that is very similar to B. anthracis ? that initiates sporulation. Thus, the Fah phage may be able to change its gene expression strategy depending on the physiological state (exponential growth versus sporulation at nutrient limitation) of the host cell. In collaboration with the group of Dr. Alexander Solonin, Puschino, Russia, we continued to analyze temporal regulation of transcription during the establishment of mobile restriction-modification systems in naïve bacterial hosts. Since restriction endonucleases encoded by such systems will kill the host cell unless enough methyltransferase that methylates host DNA and prevents endonuclease function (and thus protects the cell) is produced, different restriction-modification systems have evolved elegant means to ensure that there is a time delay in endonuclease synthesis. We characterized the molecular mechanism that ensures such a delay in a class of systems that are controlled by specialized C (control) proteins, which are homologous to phage λ repressor protein. Structure-functional analysis of RNAP Studies of RNAP-binding transcription inhibitor Microcin J25 (MccJ25) and Streptolydigin (Stl) were performed. Research on MccJ25, which functions by binding to RNAP and blocking the secondary channel--a conduit for NTP substrates to the catalytic center--focused on structure-activity analysis of this peptide antibiotic, using partially proteolyzed MccJ25 derivatives as well as genetically engineered mutants. The results allowed to delineate MccJ25 determinants responsible for cell entry and RNA polymerase binding. Research on Stl involved structural analysis (in collaboration with Dr. Dmitriy Vassylyev’s laboratory, University of Alabama, Birmingham) as well as mutational, biochemical and kinetic analysis of the inhibition process. The results lead to a view that binding of Stl greatly reduces the rate of RNA polymerase isomerization that is required to load the NTP substrate from a preinsertion site to an insertion site from which catalysis of phosphodiester bind synthesis can occur. In collaboration with Seth Darst, Rockefeller University, we developed a genetic system that allows us to produce recombinant T. aquaticus RNA polymerase for structural studies. This is a major advance as it will allow us to perform structural analysis of RNA polymerase mutants, which heretofore was impossible. As part of a long-standing collaboration with the laboratory of Dr. Martin Buck, Imperial College, London, UK, we continued the analysis of the contribution of individual RNAP core domains to transcription initiation by E. coli RNAP σ54 holoenzyme. We analyzed the role of the downstream jaw of the β‘ subunit in the izomerization of the σ54 holoenzyme from closed to open complex. Publications Phadtare S., Kazakov T., Bubunenko M., Court D. L., Pestova T, and Severinov K., Transcription Antitermination by Translation Initiation Factor IF1 J. Bacteriol. 2007 189: 4087-4093. Zenkin N, Kulbachinskiy A, Yuzenkova Y, Mustaev A, Bass I, Severinov K, Brodolin K. Region 1.2 of the RNA polymerase sigma subunit controls recognition of the -10 promoter element.   EMBO J. 2007 Feb 21;26(4):955-64. Epub 2007 Feb 1. Kazakov, T., Metlitskaya, A., and Severinov, K. (2007) Structure-activity analysis of translation inhibitor microcin C. J. Bacteriol., 189, 2114-2118. Naryshkina, T., Liu, J., Florens, L., Swanson, S. K., Inman, R., Pavlov, A. R., Pavlova, N. V., Kozyavkin, S. A., Washburn, M., Mushegian, A., and Severinov, K. (2006) Genome structure and virion composition of a large Thermus thermophilus bacteriophage fYS40. J. Mol. Biol., 364, 667-677 Baxter, K., Lee, J., Minakhin, L., Severinov, K., D. H. Hinton. (2006) Interactions of region 4 of the s70 subunit of E. coli RNA polymerase with the T4 co-activator, AsiA, and the T4 activator, MotA, needed for s70 appropriation. J. Mol. Biol., 363, 931-944 Djordjevic, M., Semenova, E., Shraiman, B., and Severinov, K. (2006) Quantitative analysis of bacteriophage Xp10 transcription strategy. Virology, 354, 240-251 Kuznedelov, K., Komissarova, N., and Severinov, K. (2006) The role of the bacterial RNA polymerase b subunit flexible flap domain in transcription termination. Dokl. Biochem. Biophys., 410, 263-266 Naryshkina, T., Kuznedelov, K., and Severinov, K.. (2006) Structure-based analysis of RNA polymerase function: the role of the largest subunit ’s lid element in the formation of extended RNA-DNA hybrid. J. Mol. Biol., 361, 634-643 Zenkin, N., Yuzenkova, Y., and Severinov, K. (2006) Transcriptional proofreading through a ribozyme-like activity of the nascent transcript. Science, 313, 518-520 Wigneshweraraj, S. R., Savalia, D., Severinov, K., and Buck, M. (2006) Collaboration between the b’ clamp and the b’ jaw domains during DNA opening by the bacterial RNA polymerase at s54-dependent promoters. J. Mol. Biol., 359, 1182-1195 Feklistov, A., Sevostyanova, A., Barinova, N., E. Heyduk, Bass, I., Vvedenskaya, I., Heyduk, E., Nikiforov, V., Heyduk, T., Severinov, K., and Kulbachinskiy, A. (2006) A novel downstream promoter element recognized by free RNA polymerase s subunit determines species-specific promoter recognition by RNA polymerase holoenzyme. Mol. Cell, 23, 97-107 Metlitskaya, A., Kazakov, T., Kommer, A., Pavlova, O., Krashenninikov, I., Kolb, V., Khmel’, I., and Severinov, K. (2006) Aspartyl-tRNA synthetase is the target of peptidenucleotide antibiotic Microcin C. J. Biol. Chem., 281, 18033-18042 Kuznedelov, K., Lamour, V., Patikoglou, G., Darst, S. A., and Severinov, K. (2006) Recombinant Thermus aquaticus RNA polymerase for structural studies. J. Mol. Biol., 359, 110-121 Severinova, E. and Severinov, K. (2006) Localization of the E. coli RNA polymerase b’ subunit residue phosphorylated by bacteriophage T7 kinase Gp0.7. J. Bacteriol., 188, 3470-3476 Heyduk, E., Kuznedelov, K., Severinov, K., and Heyduk, T. (2006) Bacterial promoter consensus adenine at position -11 of nontemplate strand is important for the nucleation of promoter melting. J. Biol. Chem., 281, 12362-12369 Cristóbal, R. E., Solbiati, J. O., Zenoff, A. M., Vincent, P., Y. Yuzenkova, Salomón, R. A., Severinov, K., and Far ías, R. N. (2006) Microcin J25 uptake: His5 of the MccJ25 lariat ring is involved in the interaction with the inner-membrane MccJ25-transporter protein SbmA. J. Bacteriol., 188, 3324-3328 Phadtare, S., Tadigotla, V., Shin, W.-H., Sengupta, A., and Severinov, K. (2006) Analysis of Escherichia coli global gene expression profiles in response to overexpression and deletion of CspC and CspE. J. Bacteriol., 188, 2521-2527 Zenkin, N., Naryshkina, T., Kuznedelov, K., and Severinov, K. (2006) The molecular mechanism of replication primer synthesis by RNA polymerase. Nature, 439, 617-620 Semenova, E., Minakhin, M., Vasilov, A., Solonin, A., Heyduk, T., Zakharova, M., and Severinov, K. (2005) Transcription regulation of the EcoRV restriction-modification system. Nucleic Acids Res., 33, 6942-6951 Minakhin, L., Semenova, E., Liu, J., Vasilov, A., Severinova, E., Gabisonia, T., Inman, R., Mushegian, A., and Severinov, K. (2005) Genome and gene expression of Bacillus anthracis bacteriophage Fah. J. Mol. Biol., 354, 1-15 Phadtare, S. and Severinov, K. (2005) Elucidation of the mechanism of nucleic acid melting by Escherichia coli CspE. Nucl. Acids. Res., 33, 5583-5590 Wigneshweraraj, S. R., Burrows, P. S., Severinov, K., and Buck, M. (2005) Stable DNA opening within open promoter complexes is mediated by the RNA polymerase b'-jaw domain. J. Biol. Chem, 280, 36176-36184 Temiakov, D., Zenkin, N., Vassylyeva, M., Tahirov, T., Anikin, M., Kashkina, E., Savkina, M., Zorov, S., Nikiforov, V., Igarashi, N., Matsugaki, N., Wakatsuki, S., Perederina, A., Severinov, K., and Vassylyev, D. (2005) Structural basis for transcription inhibition by antibiotic streptolydigin. Mol. Cell, 19, 655-666 Phadtare, S. and Severinov, K. (2005) The extended -10 motif is critical for activity of the cspA promoter but does not contribute to low-temperature transcription. J. Bacteriol., 187, 6584-6589 Sosunov, V., Zorov, S., Sosunova, E., Nikolaev, A., Bass, I., Goldfarb, A., Nikiforov, V., Severinov, K., and Mustaev, A. (2005) The role of the aspartate triad in catalytic activities of multisubunit RNA polymerase. Nucl. Acids. Res., 33, 4202-4211 Brodolin, K., Zenkin, N., and Severinov, K. (2005) Remodeling of the s70 subunit non-template DNA strand contacts during the final step of transcription initiation. J. Mol. Biol., 350, 930-937 Semenova, E., Yuzenkova, J., Peduzzi, J., Rebuffat, S., and Severinov, K. (2005) Structure-activity analysis of Microcin J25: distinct parts of the threaded lasso molecule are responsible for interaction with bacterial RNA polymerase. J. Bacteriol., 187, 3859-3863 Nickels, B. E., Garrity, S., Mekler, V., Minakhin, L., Severinov, K., Ebright, R. H., and Hochschild, A. (2005) Altering the interaction between s70 and the b-flap of E. coli RNA polymerase provides evidence for a barrier to the extension of the nascent RNA during early elongation. Proc. Natl. Acad. Sci. USA, 102, 4488-4493 Campbell, E. A., Pavlova, O., Zenkin, N., Leon, F., Irschik, H., Jansen, R., Severinov, K., and Darst, S. A. (2005) Structural, functional, and genetic analysis of sorangicin inhibition of bacterial RNA polymerase. EMBO J., 24, 674-682 Semenova, E., Djordjevic, M., Shraiman, B., and Severinov, K. (2005) The tale of two polymerases: Transcription profiling and gene expression strategy of bacteriophage Xp10. Mol. Microbiol., 55, 764-777 Lab Support Dr. Konstantin Kuznedelov, Research Associate Dr. Leonid Minakhin, Research Assistant Dr. Ekaterina Semenova, Research Assistant Yulia Yuzenkova, Research Assistant Nikolas Zenkin, Research Assistant Dr. Tatyana Naryshkina, Postdoctoral Associate Dr. Andrey Kulbachinskiy, Postdoctoral Fellow Dr. Anatoly Vassilov, Postdoctoral Fellow Ekaterina Bogdanova, Visiting scientist Timur Kazakov, Visiting Scientist Dhruti Savalia, Graduate Fellow Olga Pavlova, Visiting Undergraduate Student