Coliphage N4 transcription is carried out by three different RNA polymerases (RNAP) providing unique opportunities to study structure-function relationships in RNAPases, the role of DNA structure, supercoiling and single-stranded DNA binding (SSB) proteins in transcriptional activation. We propose to continue studies on the structure, mechanism of promoter recognition and activation of the virion-encapsidated, 320-kDa single-polypeptide RNAP (vRNAP) which is responsible for the synthesis of phage early RNAs. vRNAP recognizes a 5-7 bp stem, 3b loop hairpin and specific sequences at its promoters. N4 vRNAP promoters are activated by supercoiling and Eco SSB. Supercoiling leads to a Mg(II)- and sequence-dependent extrusion of the promoter hairpins. We have proposed that Eco SSB invades this structure with melting of the complementary strand hairpin and stabilization of the template strand hairpin which is recognized by vRNAP. We propose to characterize the DNA structural transition occurring at vRNAP promoters using fluorescence resonance transfer (FRET). We will further characterize the interaction vRNAP with its promoters by elucidating the sequence determinants of promoter binding through a binding site selection protocol and the use of a quantitative binding assay. The role of Eco SSB in providing the structure of the """"""""activated promoter"""""""" will be defined by footprinting mapping of the topology of the """"""""activated"""""""" promoter, determining the role of promoter downstream sequences and of single-strandedness in EcoSSB invasion and footprinting of vRNAP at the """"""""activated promoter"""""""". The stoichiometry of Eco SSB binding at the activated promoter will be determined by scanning force microscopy. We will characterize the role of Eco SSB and promoter sequences in promoter clearance. Finally, we will define the vRNAP domain involved in promoter recognition through proteolysis and crosslinking studies. Results of these experiments will provide insights into a new strategy of promoter-RNAP interaction, novel determinants of protein-DNA interaction, as well as the role of single-stranded DNA binding proteins in regulation of gene expression.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI012575-25
Application #
6087504
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1998-11-15
Project End
2003-10-31
Budget Start
1999-11-01
Budget End
2000-10-31
Support Year
25
Fiscal Year
2000
Total Cost
$367,440
Indirect Cost
Name
University of Chicago
Department
Genetics
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637
Lenneman, Bryan R; Rothman-Denes, Lucia B (2015) Structural and biochemical investigation of bacteriophage N4-encoded RNA polymerases. Biomolecules 5:647-67
Molodtsov, Vadim; Nawarathne, Irosha N; Scharf, Nathan T et al. (2013) X-ray crystal structures of the Escherichia coli RNA polymerase in complex with benzoxazinorifamycins. J Med Chem 56:4758-63
Basu, Ritwika S; Murakami, Katsuhiko S (2013) Watching the bacteriophage N4 RNA polymerase transcription by time-dependent soak-trigger-freeze X-ray crystallography. J Biol Chem 288:3305-11
Chen, Yuanyuan; Basu, Ritwika; Gleghorn, Michael L et al. (2011) Time-resolved events on the reaction pathway of transcript initiation by a single-subunit RNA polymerase: Raman crystallographic evidence. J Am Chem Soc 133:12544-55
Gleghorn, Michael L; Davydova, Elena K; Basu, Ritwika et al. (2011) X-ray crystal structures elucidate the nucleotidyl transfer reaction of transcript initiation using two nucleotides. Proc Natl Acad Sci U S A 108:3566-71
Yano, Sho T; Rothman-Denes, Lucia B (2011) A phage-encoded inhibitor of Escherichia coli DNA replication targets the DNA polymerase clamp loader. Mol Microbiol 79:1325-38
McPartland, Jennifer; Rothman-Denes, Lucia B (2009) The tail sheath of bacteriophage N4 interacts with the Escherichia coli receptor. J Bacteriol 191:525-32
Davydova, Elena K; Kaganman, Irene; Kazmierczak, Krystyna M et al. (2009) Identification of bacteriophage N4 virion RNA polymerase-nucleic acid interactions in transcription complexes. J Biol Chem 284:1962-70
Gleghorn, Michael L; Davydova, Elena K; Rothman-Denes, Lucia B et al. (2008) Structural basis for DNA-hairpin promoter recognition by the bacteriophage N4 virion RNA polymerase. Mol Cell 32:707-17
Murakami, Katsuhiko S; Davydova, Elena K; Rothman-Denes, Lucia B (2008) X-ray crystal structure of the polymerase domain of the bacteriophage N4 virion RNA polymerase. Proc Natl Acad Sci U S A 105:5046-51

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