RNA polymerase and the lead ribosome may be physically linked in E. coli by the two-domain NusG protein. This will be demonstrated at the biochemical, structural and genetic levels. We intend to isolate a complex between FL NusG and the 70S ribosome, and to probe its structure using cryo-EM. We will screen a number of mutants for their effects on coupling transcription and translation. Our results indicate that RelA is required for coupling and this will be explored genetically and biochemically. Our studies have implicated tmRNA as the function that uncouples transcription and translation when translation is inhibited. Structural data indicates that tmRNA can displace NusG from the ribosome;we will test this in vitro. NMR and biochemical analyses suggest that NusA can displace NusG from RNAP. This will be tested in a purified transcription system. Chromosome integrity depends on transcription termination. The details of this reaction will be explored. Finally, we find that transcription is linked to repair of a variety of DNA damages. The influence of transcription depends on the type of DNA damage. We will explore the pathways that repair DNA DSBs, DNA adducts, and interstrand crosslinks, and that can remove tightly bound protein from the DNA template.

Public Health Relevance

Transcription and translation are coordinated in all living organisms. This project focuses on how these two synthetic pathways are coupled in E. coli. The project also focuses on how transcription termination in E. coli relates to chromosome integrity and to repair of DNA damage.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
2R01GM037219-26
Application #
8759962
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Sledjeski, Darren D
Project Start
Project End
Budget Start
Budget End
Support Year
26
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
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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