Konstantin V. Severinov Professor Rutgers University Department of Molecular Biology and Biochemistry Waksman Institute of Microbiology 190 Frelinghuysen Rd.. Room 224 Piscataway. NJ 08855-0759 (732) 445 6095 FAX -5735 severik@waksman.rutgers.edu

Genetic and biochemical analysis of RNA polymerases from E. coli and yeast. site-directed modification of proteins

Transcription. the first step. and a major regulatory checkpoint of gene expression is carried out by DNA-dependent RNA polymerases. RNA polymerase alone or in complex with regulatory factors is central to all steps of transcription. Defective transcription is the cause of aberrant growth and development and may result in malignant transformation. Our long-term scientific goal is to understand transcription mechanism and regulation in molecular detail.

One project in this laboratory concerns domain organization of the two largest subunits (ß' and ß) of RNA polymerase from Escherichia coli. Our goal is to use a combination of biochemical. genetic. chemical and structural approaches to bridge the gap between the primary RNA polymerase sequence. available functional data. and the three-dimensional model of RNA polymerase. We have showed that 25% of the RNA polymerase ß subunit sequence is dispensable and could be deleted without affecting basic function. A principal result was the demonstration that dispensable regions could be involved in interactions with transcription factors. We also demonstrated that both ß' and ß can be physically split without preventing RNA polymerase assembly and function. These results showed that RNA polymerase is a highly modular enzymes and opened several new avenues of research which are currently being pursued. The assembly-competent subunit fragments are being used to investigate intersubunit interactions during RNA polymerase assembly. Split RNA polymerases are also being used to map chemical crosslinks between RNA polymerase and derivatized nascent RNA or DNA template.

This work is now also being extended to RNA polymerase I (pol I) from yeast. We use a unique genetic system that makes pol I dispensable for cell viability to uncover structure-functional relationships of this enzyme. In addition. we use pol I as a vehicle to assemble in vivo chimeric RNA polymerases,harboring domain swaps between pol I and pol II and pol III.

Finally. in collaboration with a chemistry lab we are designing a general method of site-specific. chemical modification of proteins. We developed a very important technique that allows us to incorporate fluorescent and crosslinkable labels within ca. 50 C-terminal amino acids of a protein. Our approach involves an in vitro ligation of the smaller. chemically synthesized C-terminal fragment of a protein to the larger. recombinant N-terminal fragment which is genetically fused to protein self-splicing element intein. At conditions favoring intein excision and in the presence of the C-terminal fragment containing N-terminal cysteine efficient ligation of the N and the C-terminal segments is achieved. Our immediate plans are to use this system to systematically study protein-protein and protein-nucleic acids interactions in transcription complexes.

Selected Publications

Phadtare S, Kazakov T, Bubunenko M, Court DL, Pestova T, Severinov K. (2007) Transcription antitermination by translation initiation Factor IF1. J Bacteriol. 189(11):4087-93.

Dailidiene D, Tan S, Ogura K, Zhang M, Lee AH, Severinov K, Berg DE. (2007) Urea sensitization caused by separation of Helicobacter pylori RNA polymerase beta and beta' subunits. Helicobacter. 12(2):103-11.

Zenkin N, Kulbachinskiy A, Yuzenkova Y, Mustaev A, Bass I, Severinov K, Brodolin K.(2007) Region 1.2 of the RNA polymerase sigma subunit controls recognition of the -10 promoter element. EMBO J. 26(4):955-64.

Sevostyanova A, Djordjevic M, Kuznedelov K, Naryshkina T, Gelfand MS, Severinov K, Minakhin L. (2007) Temporal regulation of viral transcription during development of Thermus thermophilus bacteriophage phiYS40. J Mol Biol. 366(2):420-35.

Kazakov T, Metlitskaya A, Severinov K. (2007) Amino acid residues required for maturation, cell uptake, and processing of translation inhibitor microcin C. J Bacteriol. 189(5):2114-8.

Naryshkina T, Liu J, Florens L, Swanson SK, Pavlov AR, Pavlova NV, Inman R, Minakhin L, Kozyavkin SA, Washburn M, Mushegian A, Severinov K. (2006) Thermus thermophilus bacteriophage phiYS40 genome and proteomic characterization of virions. J Mol Biol. 364(4):667-77.

Baxter K, Lee J, Minakhin L, Severinov K, Hinton DM. (2006) Mutational analysis of sigma70 region 4 needed for appropriation by the bacteriophage T4 transcription factors AsiA and MotA. J Mol Biol. 363(5):931-44.

Djordjevic M, Semenova E, Shraiman B, Severinov K. (2006) Quantitative analysis of a virulent bacteriophage transcription strategy. Virology. 354(2):240-51.

Zenkin N, Yuzenkova Y, Severinov K. (2006) Transcript-assisted transcriptional proofreading.
Science. 313(5786):518-20.

Feklistov A. Barinova N. Sevostyanova A. Heyduk E. Bass I. Vvedenskaya I. Kuznedelov K. Merkiene E. Stavrovskaya E. Klimasauskas S. Nikiforov V. Heyduk T. Severinov K. Kulbachinskiy A. (2006) A basal promoter element recognized by free RNA Polymerase sigma subunit determines promoter recognition by RNA Polymerase Holoenzyme. Mol Cell. 23(1):97-107.

Naryshkina T. Kuznedelov K. Severinov K. (2006) The role of the largest RNA Polymerase subunit lid element in preventing the formation of extended RNA-DNA hybrid. J Mol Biol. 361(4):634-43.

Wigneshweraraj SR. Savalia D. Severinov K. Buck M. (2006) Interplay between the beta' Clamp and the beta' Jaw domains during DNA opening by the bacterial RNA Polymerase at sigma(54)-dependent promoters. J Mol Biol. 359(5):1182-95.

Severinova E. Severinov K. (2006) Localization of the Escherichia coli RNA polymerase beta' subunit residue phosphorylated by bacteriophage T7 kinase Gp0.7. J Bacteriol. 188(10):3470-6.

de Cristobal RE. Solbiati JO. Zenoff AM. Vincent PA. Salomon RA. Yuzenkova J. Severinov K. Farias RN. (2006) Microcin J25 uptake: His5 of the MccJ25 lariat ring is involved in interaction with the inner membrane MccJ25 transporter protein SbmA. J Bacteriol. 188(9):3324-8.

Kuznedelov K. Lamour V. Patikoglou G. Chlenov M. Darst SA. Severinov K. (2006) Recombinant Thermus aquaticus RNA polymerase for structural studies. J Mol Biol. 359(1):110-21. E

Metlitskaya A. Kazakov T. Kommer A. Pavlova O. Praetorius-Ibba M. Ibba M. Krasheninnikov I. Kolb V. Khmel I. Severinov K. (2006) Aspartyl-tRNA Synthetase Is the target of peptide nucleotide antibiotic Microcin C. J Biol Chem. 281(26):18033-42.

Phadtare S. Tadigotla V. Shin WH. Sengupta A. Severinov K. (2006) Analysis of Escherichia coli global gene expression profiles in response to overexpression and deletion of CspC and CspE. J Bacteriol. 188(7):2521-7.

Heyduk E. Kuznedelov K. Severinov K. Heyduk T. (2006) A consensus adenine at position -11 of the nontemplate strand of bacterial promoter is important for nucleation of promoter melting.J Biol Chem. 281(18):12362-9.

Zenkin N. Naryshkina T. Kuznedelov K. Severinov K. (2006) The mechanism of DNA replication primer synthesis by RNA polymerase. Nature. 439(7076):617-20.

Semenova E. Minakhin L. Bogdanova E. Nagornykh M. Vasilov A. Heyduk T. Solonin A. Zakharova M. Severinov K. (2005) Transcription regulation of the EcoRV restriction-modification system. Nucleic Acids Res. 33(21):6942-51.

Minakhin L. Semenova E. Liu J. Vasilov A. Severinova E. Gabisonia T. Inman R. Mushegian A. Severinov K. (2005) Genome sequence and gene expression of Bacillus anthracis bacteriophage Fah. J Mol Biol. 354(1):1-15.

Phadtare S. Severinov K. (2005) Nucleic acid melting by Escherichia coli CspE. Nucleic Acids Res. 33(17):5583-90.

Temiakov D. Zenkin N. Vassylyeva MN. Perederina A. Tahirov TH. Kashkina E. Savkina M. Zorov S. Nikiforov V. Igarashi N. Matsugaki N. Wakatsuki S. Severinov K. Vassylyev DG. (2005) Structural basis of transcription inhibition by antibiotic streptolydigin. Mol Cell. 19(5):655-66.

Phadtare S. Severinov K. (2005) Extended -10 motif is critical for activity of the cspA promoter but does not contribute to low-temperature transcription. J Bacteriol. 187(18):6584-9.

Wigneshweraraj SR. Burrows PC. Severinov K. Buck M. (2005) Stable DNA opening within open promoter complexes is mediated by the RNA polymerase beta'-jaw domain. J Biol Chem. 280(43):36176-84.

Sosunov V. Zorov S. Sosunova E. Nikolaev A. Zakeyeva I. Bass I. Goldfarb A. Nikiforov V. Severinov K. Mustaev A. (2005) The involvement of the aspartate triad of the active center in all catalytic activities of multisubunit RNA polymerase. Nucleic Acids Res. ;33(13):4202-11.

Brodolin K. Zenkin N. Severinov K. (2005) Remodeling of the sigma70 subunit non-template DNA strand contacts during the final step of transcription initiation. J Mol Biol. 350(5):930-7.

Semenova E. Yuzenkova Y. Peduzzi J. Rebuffat S. Severinov K. (2005) Structure-activity analysis of microcinJ25: distinct parts of the threaded lasso molecule are responsible for interaction with bacterial RNA polymerase. J Bacteriol. 187(11):3859-63.

Minakhin L. Severinov K. (2005) Transcription regulation by bacteriophage T4 AsiA. Protein Expr Purif. 41(1):1-8.

Nickels BE. Garrity SJ. Mekler V. Minakhin L. Severinov K. Ebright RH. Hochschild A. (2005) The interaction between sigma70 and the beta-flap of Escherichia coli RNA polymerase inhibits extension of nascent RNA during early elongation. Proc Natl Acad Sci U S A. 102(12):4488-93.

Campbell EA. Pavlova O. Zenkin N. Leon F. Irschik H. Jansen R. Severinov K. Darst SA. (2005) Structural. functional. and genetic analysis of sorangicin inhibition of bacterial RNA polymerase. EMBO J. 24(4):674-82.

Semenova E. Djordjevic M. Shraiman B. Severinov K. (2005) The tale of two RNA polymerases: transcription profiling and gene expression strategy of bacteriophage Xp10. Mol Microbiol. 55(3):764-77.

Budarina ZI. Nikitin DV. Zenkin N. Zakharova M. Semenova E. Shlyapnikov MG. Rodikova EA. Masyukova S. Ogarkov O. Baida GE. Solonin AS. Severinov K. (2004) A new Bacillus cereus DNA-binding protein. HlyIIR. negatively regulates expression of B. cereus haemolysin II. Microbiology. 150(Pt 11):3691-701.

Burrows PC. Severinov K. Buck M. Wigneshweraraj SR. (2004) Reorganisation of an RNA polymerase-promoter DNA complex for DNA melting. EMBO J. 23(21):4253-63.

Wigneshweraraj SR. Burrows PC. Nechaev S. Zenkin N. Severinov K. Buck M. (2004) Regulated communication between the upstream face of RNA polymerase and the beta' subunit jaw domain. EMBO J. 23(21):4264-74.

King RA. Markov D. Sen R. Severinov K. Weisberg RA. (2004) A conserved zinc binding domain in the largest subunit of DNA-dependent RNA polymerase modulates intrinsic transcription termination and antitermination but does not stabilize the elongation complex. J Mol Biol. 342(4):1143-54.

Markov D. Christie GE. Sauer B. Calendar R. Park T. Young R. Severinov K. (2004) P2 growth restriction on an rpoC mutant is suppressed by alleles of the Rz1 homolog lysC. J Bacteriol. 186(14):4628-37.

Adelman K. Yuzenkova J. La Porta A. Zenkin N. Lee J. Lis JT. Borukhov S. Wang MD. Severinov K. (2004) Molecular mechanism of transcription inhibition by peptide antibiotic Microcin J25. Mol Cell. 14(6):753-62.

Zenkin N. Severinov K. (2004) The role of RNA polymerase sigma subunit in promoter-independent initiation of transcription. Proc Natl Acad Sci U S A. 101(13):4396-400.

Phadtare S. Inouye M. Severinov K. (2004) The mechanism of nucleic acid melting by a CspA family protein. J Mol Biol. 337(1):147-55.

Phadtare S. Severinov K. Inouye M. (2003)Assay of transcription antitermination by proteins of the CspA family. Methods Enzymol. 371:460-71.

Mustaev A. Zaychikov E. Grachev M. Kozlov M. Severinov K. Epshtein V. Korzheva N. Bereshchenko O. Markovtsov V. Lukhtanov E. Tsarev I. Maximova T. Kashlev M. Bass I. Nikiforov V. Goldfarb A. (2003) Strategies and methods of cross-linking of RNA polymerase active center. Methods Enzymol. 371:191-206.

Wigneshweraraj SR. Nechaev S. Bordes P. Jones S. Cannon W. Severinov K. Buck M. (2003) Enhancer-dependent transcription by bacterial RNA polymerase: the beta subunit downstream lobe is used by sigma 54 during open promoter complex formation. Methods Enzymol. 370:646-57.

Nechaev S. Imburgio D. Severinov K. (2003) Purification and characterization of bacteriophage-encoded inhibitors of host RNA polymerase: T-odd phage gp2-like proteins. Methods Enzymol. 370:212-25.

Kuznedelov K. Minakhin L. Severinov K. (2003) Preparation and characterization of recombinant Thermus aquaticus RNA polymerase. Methods Enzymol. 370:94-108.

Zakharova M. Minakhin L. Solonin A. Severinov K. (2004) Regulation of RNA polymerase promoter selectivity by covalent modification of DNA. J Mol Biol. 335(1):103-11.

Naryshkina T. Bruning A. Gadal O. Severinov K. (2003) Role of second-largest RNA polymerase I subunit Zn-binding domain in enzyme assembly. Eukaryot Cell. 2(5):1046-52.

Phadtare S. Hwang J. Severinov K. Inouye M. (2003) CspB and CspL. thermostable cold-shock proteins from Thermotoga maritima. Genes Cells. 8(10):801-10.

Wilson KA. Kalkum M. Ottesen J. Yuzenkova J. Chait BT. Landick R. Muir T. Severinov K. Darst SA. (2003) Structure of microcin J25. a peptide inhibitor of bacterial RNA polymerase. is a lassoed tail. J Am Chem Soc. 125(41):12475-83.