The machinery responsible for making proteins (e.g. ribosomal RNA, ribosomal proteins, translation factors, and tRNAs) is central to growth and development of all organisms. The control of its synthesis has been a central issue in molecular microbiolgy for almost 60 years. It had become clear in previous project periods that our work was central not only for understanding ribosome synthesis but also for understanding the mechanism of transcription initiation in general. What has become apparent in the last few years is that the systems that regulate rRNA promoters also directly regulate a wide variety of other promoters as well, many of which were not anticipated because their connection to the protein synthetic apparatus was not apparent. However, we now know that the molecules that regulate rRNA promoters are utilized throughout the bacterial kingdom, they play crucial roles in virulence and infectious disease, and they contribute to genome maintenance as well as to transcription. In the next project period, we will (i) continue our studies on the DNA-RNAP interactions that contribute to the transition from a closed to an open complex in the promoter mechanism, (ii) explore the role of specific promoter regions in defining the transcription start site, (iii) continue our examination of the control of transcription by ppGpp and DksA by defining their binding sites on RNA polymerase and determining their mechanism of action, (iv) expand our analysis of control by these molecules to additional promoters in order to understand what differentiates positively from negatively regulated targets, (v) continue studying the mechanism of action ofthe RNA polymerase assembly factor CrI, and (vi) continue studies on our recent discovery that E. coli makes a structure analogous to the eukaryotic nucleolus. We will expand these studies to explore other potential long-range interactions between distant parts of the bacterial chromosome.

Public Health Relevance

It has become apparent in the lastfew years that the systems that regulate rRNA promoters also directly regulate a wide variety of other promoters as well. Some of these factors play crucial roles in virulence and infectious disease, as well as contributing to gene expression and the fidelity of replication. Thus, understanding these fundamental processes has the potential to impact human health directly.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM037048-28
Application #
8691848
Study Section
No Study Section (in-house review) (NSS)
Program Officer
Sledjeski, Darren D
Project Start
1988-07-01
Project End
2017-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
28
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Microbiology/Immun/Virology
Type
Earth Sciences/Resources
DUNS #
City
Madison
State
WI
Country
United States
Zip Code
53715
Winkelman, Jared T; Vvedenskaya, Irina O; Zhang, Yuanchao et al. (2016) Multiplexed protein-DNA cross-linking: Scrunching in transcription start site selection. Science 351:1090-3
Gaal, Tamas; Bratton, Benjamin P; Sanchez-Vazquez, Patricia et al. (2016) Colocalization of distant chromosomal loci in space in E. coli: a bacterial nucleolus. Genes Dev 30:2272-2285
Winkelman, Jared T; Chandrangsu, Pete; Ross, Wilma et al. (2016) Open complex scrunching before nucleotide addition accounts for the unusual transcription start site of E. coli ribosomal RNA promoters. Proc Natl Acad Sci U S A 113:E1787-95
Ross, Wilma; Sanchez-Vazquez, Patricia; Chen, Albert Y et al. (2016) ppGpp Binding to a Site at the RNAP-DksA Interface Accounts for Its Dramatic Effects on Transcription Initiation during the Stringent Response. Mol Cell 62:811-23
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Gyorfy, Zsuzsanna; Draskovits, Gabor; Vernyik, Viktor et al. (2015) Engineered ribosomal RNA operon copy-number variants of E. coli reveal the evolutionary trade-offs shaping rRNA operon number. Nucleic Acids Res 43:1783-94
Winkelman, Jared T; Winkelman, Bradford T; Boyce, Julian et al. (2015) Crosslink Mapping at Amino Acid-Base Resolution Reveals the Path of Scrunched DNA in Initial Transcribing Complexes. Mol Cell 59:768-80
Yang, Yun; Darbari, Vidya C; Zhang, Nan et al. (2015) TRANSCRIPTION. Structures of the RNA polymerase-σ54 reveal new and conserved regulatory strategies. Science 349:882-5

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