Abstract

Bacteriophage N4-infected E. coli show rifampicin and streptolydigin resistant transcription even when the drugs are added prior to infection. All N4 transcription is rifampicin resistant and therefore independent of the host RNA polymerase. Analysis of transcription during N4 infection has revealed the presence of two new rifampicin-resistant RNA polymerizing activities. The first activity is unprecedented in that its appearance does not require transcription or translation of the phage genome after infection. This activity is virion encapsulated, injected into the cell with the DNA and transcribes viral early genes. The second activity requires the proper expression and function of two N4 genes for its appearance and is responsible for the transcription of late N4 genes. Genetic studies have demonstrated that both activities are required for viral transcription and, moreover, that the virion encapsulated RNA polymerase is required directly for DNA replication. We propose to undertake a full biochemical and genetic characterization of both transcribing activities. Related to this aim will be the obtention of a restriction endonuclease map of N4 DNA to be used to map and isolate promoters for the N4 RNA polymerases. Finally, an in vitro system, capable of supporting N4 DNA replication, will be developed to study the role of the virion-associated RNA polymerase in N4-DNA synthesis.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI012575-11
Application #
3125224
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1978-05-01
Project End
1988-04-30
Budget Start
1985-05-01
Budget End
1986-04-30
Support Year
11
Fiscal Year
1985
Total Cost
Indirect Cost

  Institution

Name
University of Chicago
Department
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637

  Publications

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

Showing the most recent 10 out of 41 publications