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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Sledjeski, Darren D
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University of Wisconsin Madison
Schools of Earth Sciences/Natur
United States
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Winkelman, Jared T; Gourse, Richard L (2017) Open complex DNA scrunching: A key to transcription start site selection and promoter escape. Bioessays 39:
Girard, Mary E; Gopalkrishnan, Saumya; Grace, Elicia D et al. (2017) DksA and ppGpp Regulate the ?S Stress Response by Activating Promoters for the Small RNA DsrA and the Anti-Adapter Protein IraP. J Bacteriol :
Burgos, Hector L; O'Connor, Kevin; Sanchez-Vazquez, Patricia et al. (2017) Roles of Transcriptional and Translational Control Mechanisms in Regulation of Ribosomal Protein Synthesis in Escherichia coli. J Bacteriol 199:
Gopalkrishnan, Saumya; Ross, Wilma; Chen, Albert Y et al. (2017) TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp. Proc Natl Acad Sci U S A 114:E5539-E5548
Cuthbert, Bonnie J; Ross, Wilma; Rohlfing, Amy E et al. (2017) Dissection of the molecular circuitry controlling virulence in Francisella tularensis. Genes Dev 31:1549-1560
Maouche, Rim; Burgos, Hector L; My, Laetitia et al. (2016) Coexpression of Escherichia coli obgE, Encoding the Evolutionarily Conserved Obg GTPase, with Ribosomal Proteins L21 and L27. J Bacteriol 198:1857-1867
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
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
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 62:811-823

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