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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Sledjeski, Darren D
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Columbia University (N.Y.)
Schools of Medicine
New York
United States
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Saxena, Shivalika; Myka, Kamila K; Washburn, Robert et al. (2018) Escherichia coli transcription factor NusG binds to 70S ribosomes. Mol Microbiol 108:495-504
Gottesman, Max E; Mustaev, Arkady (2018) Inorganic phosphate, arsenate, and vanadate enhance exonuclease transcript cleavage by RNA polymerase by 2000-fold. Proc Natl Acad Sci U S A 115:2746-2751
Zuber, Philipp K; Hahn, Lukas; Reinl, Anne et al. (2018) Structure and nucleic acid binding properties of KOW domains 4 and 6-7 of human transcription elongation factor DSIF. Sci Rep 8:11660
Schrank, Benjamin R; Aparicio, Tomas; Li, Yinyin et al. (2018) Nuclear ARP2/3 drives DNA break clustering for homology-directed repair. Nature 559:61-66
Kang, Jin Young; Olinares, Paul Dominic B; Chen, James et al. (2017) Structural basis of transcription arrest by coliphage HK022 Nun in an Escherichia coli RNA polymerase elongation complex. Elife 6:
Mustaev, Arkady; Roberts, Jeffrey; Gottesman, Max (2017) Transcription elongation. Transcription 8:150-161
Strauß, Martin; Vitiello, Christal; Schweimer, Kristian et al. (2016) Transcription is regulated by NusA:NusG interaction. Nucleic Acids Res 44:5971-82
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
Washburn, Robert S; Gottesman, Max E (2015) Regulation of transcription elongation and termination. Biomolecules 5:1063-78
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

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