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
Drogemuller, Johanna; Stegmann, Christian M; Mandal, Angshuman et al. (2013) An autoinhibited state in the structure of Thermotoga maritima NusG. Structure 21:365-75
Mooney, Rachel Anne; Schweimer, Kristian; Rosch, Paul et al. (2009) Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators. J Mol Biol 391:341-58
Prasch, Stefan; Jurk, Marcel; Washburn, Robert S et al. (2009) RNA-binding specificity of E. coli NusA. Nucleic Acids Res 37:4736-42
Uc-Mass, Augusto; Khodursky, Arkady; Brown, Lewis et al. (2008) Overexpression of phage HK022 Nun protein is toxic for Escherichia coli. J Mol Biol 380:812-9
Burmann, Bjorn M; Uc-Mass, Augusto; Schweimer, Kristian et al. (2008) The Y39A mutation of HK022 Nun disrupts a boxB interaction but preserves termination activity. Biochemistry 47:7335-41
Cardinale, Christopher J; Washburn, Robert S; Tadigotla, Vasisht R et al. (2008) Termination factor Rho and its cofactors NusA and NusG silence foreign DNA in E. coli. Science 320:935-8
Kim, Hyeong C; Washburn, Robert S; Gottesman, Max E (2006) Role of E.coli NusA in phage HK022 Nun-mediated transcription termination. J Mol Biol 359:10-21
Washburn, Robert S; Court, Donald L; Gottesman, Max E (2006) Role of an RNase III binding site in transcription termination at lambda nutL by HK022 Nun protein. J Bacteriol 188:6824-31
Kim, Hyeong C; Gottesman, Max E (2004) Transcription termination by phage HK022 Nun is facilitated by COOH-terminal lysine residues. J Biol Chem 279:13412-7
Kim, Hyeong C; Zhou, Jian-guang; Wilson, Helen R et al. (2003) Phage HK022 Nun protein represses translation of phage lambda N (transcription termination/translation repression). Proc Natl Acad Sci U S A 100:5308-12

Showing the most recent 10 out of 28 publications