Our project is to probe the processes that assemble various proteins and RNA to form a transcriptionally active complex. The protein-protein and RNA-protein interactions will be explored in a system that is uniquely approachable genetically and biochemically, namely E. coli and coliphage. We are focused on the mechanism of transcription termination and its suppression (antitermination). We will study two phage proteins, HK022 Nun termination factor and ?N antitermination factor. Both utilize host proteins, NusA, NusB, NusE (S10) and NusG to support their reactions. Nun and ?N bind ??RNA and nucleate a complex with the four Nus Factors and RNAP. The Nus factors are also active in suppressing transcription termination in ribosomal RNA operons. The project takes advantage of our recent isolation of strains that can support deletions in NusA, NusG, or NusB. We also propose to isolate a strain bearing a transcriptionally-inactive NusE. Informed by new structures of the Nus factors and the phage proteins, we will generate mutant proteins likely to affect their transcriptional activity. We shall interrogate individual domains in order to relate structure and function. The mutants will be tested in vivo and in a variety of in vitro assays, including purified transcription systems, RNA binding tests and structural analyses. The fundamental similarity between this prokaryotic system and eukaryotic transcription systems imply that the results obtained here will have broad biological application.

Public Health Relevance

Regulation of transcription termination is important in both prokaryotic and eukaryotic organisms. This project focuses on how transcription termination in E. coli is essential for chromosome integrity and how it relates to ribosome release.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037219-25
Application #
8515438
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Sledjeski, Darren D
Project Start
1986-07-01
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
25
Fiscal Year
2013
Total Cost
$382,870
Indirect Cost
$144,032
Name
Columbia University (N.Y.)
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Mustaev, Arkady; Vitiello, Christal L; Gottesman, Max E (2016) Probing the structure of Nun transcription arrest factor bound to RNA polymerase. Proc Natl Acad Sci U S A 113:8693-8
Strauß, Martin; Vitiello, Christal; Schweimer, Kristian et al. (2016) Transcription is regulated by NusA:NusG interaction. Nucleic Acids Res 44:5971-82
Washburn, Robert S; Gottesman, Max E (2015) Regulation of transcription elongation and termination. Biomolecules 5:1063-78
Vitiello, Christal L; Kireeva, Maria L; Lubkowska, Lucyna et al. (2014) Coliphage HK022 Nun protein inhibits RNA polymerase translocation. Proc Natl Acad Sci U S A 111:E2368-75
Vitiello, Christal L; Gottesman, Max E (2014) Bacteriophage HK022 Nun protein arrests transcription by blocking lateral mobility of RNA polymerase during transcription elongation. Bacteriophage 4:e32187
Bubunenko, Mikhail; Court, Donald L; Al Refaii, Abdalla et al. (2013) Nus transcription elongation factors and RNase III modulate small ribosome subunit biogenesis in Escherichia coli. Mol Microbiol 87:382-93
Drogemuller, Johanna; Stegmann, Christian M; Mandal, Angshuman et al. (2013) An autoinhibited state in the structure of Thermotoga maritima NusG. Structure 21:365-75
Turchiano, Michael; Sweat, Victoria; Fierman, Arthur et al. (2012) Obesity, metabolic syndrome, and insulin resistance in urban high school students of minority race/ethnicity. Arch Pediatr Adolesc Med 166:1030-6
Tran, Lillian; van Baarsel, Joshua A; Washburn, Robert S et al. (2011) Single-gene deletion mutants of Escherichia coli with altered sensitivity to bicyclomycin, an inhibitor of transcription termination factor Rho. J Bacteriol 193:2229-35
Washburn, Robert S; Gottesman, Max E (2011) Transcription termination maintains chromosome integrity. Proc Natl Acad Sci U S A 108:792-7

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