RNA polymerase (RNAP) pausing and termination are important components of gene expression in all organisms. NusA and NusG are two general transcription elongation factors that are capable of stimulating pausing and termination in bacteria. Pausing allows synchronization RNAP position with RNA folding and/or regulatory factor binding. Intrinsic and Rho-dependent termination are two transcription termination mechanisms identified in bacteria. Canonical intrinsic terminators consist of an uninterrupted RNA hairpin followed by a U-tract. Although NusA was known to stimulate intrinsic termination in vitro, since NusA is essential for viability its role on termination in vivo was not known until recently. RNA-seq studies with a B. subtilis NusA depletion strain identified a class of intrinsic terminator that requires NusA. NusA-dependent terminators have weak RNA hairpins and/or poor U-tracts. There is also evidence that NusG stimulates termination of mycobacterial RNAP at suboptimal intrinsic terminators in vitro. In Rho-dependent termination, Rho promotes transcript release when it catches up to paused RNAP. E. coli NusG participates in some Rho- dependent termination events by serving as a bridge between RNAP and Rho. NusA and NusG cooperatively stimulate pausing at two sites in the 5'UTR of the B. subtilis trp operon. NusG makes sequence-specific contacts with the non-template DNA (ntDNA) strand within the paused transcription bubble. As RNAP and template DNA must move with respect to one another for elongation to resume, interaction of NusG with both components inhibits elongation. The T-rich ntDNA sequence at the two pause sites constitutes a conserved NusG recognition motif. NET-seq will be used to identify pause sites throughout the B. subtilis genome that respond to NusA and/or NusG. The ability to deplete NusA and delete nusG without growth defects makes B. subtilis the ideal organism for these studies. By combining RNase footprinting with NET-seq (RNET-seq), the effect of NusA and NusG on the translocation state of RNAP will be determined at each pause site. Similarly, a comprehensive genomic analysis of the effects of NusA, NusG and Rho on termination in B. subtilis will be performed using strains containing all combinations of NusA depletion, ?nusG and ?rho alleles. A subset of regulatory pause sites and terminators will then be characterized in vitro. A hallmark of transcription attenuation mechanisms is the presence of overlapping antiterminator and terminator structures that form in the 5'UTR. The 5'UTR of B. subtilis yxjB contains two such sets of overlapping structures. A model will be tested in which YxjB autoregulates its expression by binding to its 5'UTR and promoting termination at both terminators by preventing formation of the two antiterminators. The model also posits that the downstream terminator hairpin sequesters the yxjB ribosome binding site. Thus, this hairpin would repress translation of transcripts that fail to terminate. A combination of in vivo expression, in vitro transcription and in vitro binding studies will be used to elucidate these complex regulatory mechanisms.

Public Health Relevance

Insight into transcription elongation mechanisms will be sought through studies of RNAP pausing, transcription termination and transcription attenuation in Bacillus subtilis. Proper regulation of gene expression is of paramount importance for the function of cells in all organisms. As several human diseases such as cancer arise in part through inappropriate gene expression, while expression of HIV and certain oncogenes are regulated by attenuation, these studies will contribute to improving human health by providing a detailed mechanistic understanding of how transcription elongation is controlled.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM098399-06
Application #
9420618
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Sledjeski, Darren D
Project Start
2012-08-01
Project End
2021-01-31
Budget Start
2018-02-01
Budget End
2019-01-31
Support Year
6
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
003403953
City
University Park
State
PA
Country
United States
Zip Code
16802
Babitzke, Paul; O'Connor, Michael (2017) Noncanonical Translation Initiation Comes of Age. J Bacteriol 199:
Mondal, Smarajit; Yakhnin, Alexander V; Babitzke, Paul (2017) Modular Organization of the NusA- and NusG-Stimulated RNA Polymerase Pause Signal That Participates in the Bacillus subtilis trp Operon Attenuation Mechanism. J Bacteriol 199:
Mondal, Smarajit; Yakhnin, Alexander V; Sebastian, Aswathy et al. (2016) NusA-dependent transcription termination prevents misregulation of global gene expression. Nat Microbiol 1:15007
Yakhnin, Alexander V; Murakami, Katsuhiko S; Babitzke, Paul (2016) NusG Is a Sequence-specific RNA Polymerase Pause Factor That Binds to the Non-template DNA within the Paused Transcription Bubble. J Biol Chem 291:5299-308
Chowdhury, Nityananda; Kwan, Brian W; McGibbon, Louise C et al. (2016) Toxin MqsR cleaves single-stranded mRNA with various 5' ends. Microbiologyopen 5:370-7
Mukherjee, Sampriti; Oshiro, Reid T; Yakhnin, Helen et al. (2016) FliW antagonizes CsrA RNA binding by a noncompetitive allosteric mechanism. Proc Natl Acad Sci U S A 113:9870-5
Yakhnin, Helen; Yakhnin, Alexander V; Babitzke, Paul (2015) Ribosomal protein L10(L12)4 autoregulates expression of the Bacillus subtilis rplJL operon by a transcription attenuation mechanism. Nucleic Acids Res 43:7032-43
Yakhnin, Alexander V; Babitzke, Paul (2014) NusG/Spt5: are there common functions of this ubiquitous transcription elongation factor? Curr Opin Microbiol 18:68-71