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 lN antitermination factor. Both utilize host proteins, NusA, NusB, NusE (S10) and NusG to support their reactions. Nun and lN bind l 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 E. coli viruses regulate termination using a mechanism that is similar to that of HIV.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
High Priority, Short Term Project Award (R56)
Project #
2R56GM037219-22A1
Application #
7916903
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Tompkins, Laurie
Project Start
1986-07-01
Project End
2010-08-31
Budget Start
2009-09-01
Budget End
2010-08-31
Support Year
22
Fiscal Year
2009
Total Cost
$398,995
Indirect Cost
Name
Columbia University (N.Y.)
Department
Biochemistry
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
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
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
Schweimer, Kristian; Prasch, Stefan; Sujatha, Pagadala Santhanam et al. (2011) NusA interaction with the ? subunit of E. coli RNA polymerase is via the UP element site and releases autoinhibition. Structure 19:945-54
Burmann, Bjorn M; Luo, Xiao; Rosch, Paul et al. (2010) Fine tuning of the E. coli NusB:NusE complex affinity to BoxA RNA is required for processive antitermination. Nucleic Acids Res 38:314-26
Burmann, Bjorn M; Schweimer, Kristian; Luo, Xiao et al. (2010) A NusE:NusG complex links transcription and translation. Science 328:501-4
Mooney, Rachel Anne; Schweimer, Kristian; Rösch, 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