Abstract

Regulation of gene expression at the level of transcription termination is ubiquitous in nature. Nevertheless the precise mechanism of the termination reaction, even in bacteria, is only partially understood. Coliphage HK022 Nun protein binds nascent phage lambda transcript at specific sites (nut) and induces transcription arrest in vitro and termination in vivo. Interactions between Nun and RNA polymerase and host factors NusA, NusB, NusE and NusG are involved in this reaction. Host Mfd protein releases Nun-arrested RNA polymerase in vitro and in vivo. We will explore the possibility that Nun interacts with a host factor analogous to NusA Nun intramolecular contacts as well as interactions between Nun and host proteins will be defined by chemical and genetic approaches. Cysteine-substituted Nun will be derivatized with cross-linkers and reacted with RNA polymerase and Nus factors. The location of the cross-link will be determined by Mass Spectrometry. Cysteine-substituted Nun will also be used to determine the location of Nun in a Nun-RNA polymerase complex by FRET analysis. Nun mutants will be characterized with respect to their interactions with RNA polymerase and NusA. Binding of Nun and the related lambda protein, N, to nut and to the RNaseIII target site in the lambda pL transcript will be studied in vivo and in vitro. The basis of Nun toxicity to E. coli will be investigated and the possibility that Nun recognizes sequences other than nut will be tested.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037219-21
Application #
7317809
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Tompkins, Laurie
Project Start
1986-07-01
Project End
2009-08-31
Budget Start
2007-12-01
Budget End
2009-08-31
Support Year
21
Fiscal Year
2008
Total Cost
$452,984
Indirect Cost

  Institution

Name
Columbia University (N.Y.)
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032

  Publications

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

Showing the most recent 10 out of 46 publications