Abstract: We use Escherichia coli as a model organism to define the mechanisms of gene regulation in bacteria. Regulation of gene expression is a critical aspect of bacterial pathogenesis, and much of our understanding has come from studies of E. coli. The vast majority of work on gene expression has focused on protein regulators, but recent studies in eukaryotes have identified thousands of non-coding RNAs (ncRNAs), many of which have regulatory functions. We have recently identified over 1,000 novel transcripts in E. coli that initiate within protein-coding genes, on both DNA strands. We have strong evidence that many of these intragenic RNAs (iRNAs) have a regulatory function. Discovery of pervasive transcription of iRNAs fundamentally changes the established view of bacterial transcriptomes. The goal of this proposal is to determine whether pervasive iRNA transcription is an important, widespread, regulatory mechanism in bacteria, analogous to microRNAs in eukaryotes. To achieve this goal we will use a multidisciplinary approach to (i) comprehensively identify iRNAs, (ii) determine the mechanisms of regulation for key selected iRNA examples, (iii) determine the impact of gene regulation by iRNAs on virulence in a pathogenic strain of E. coli. We are ideally prepared to undertake this project due to my expertise and training in bacterial genetics and genomics, and my willingness to challenge long-standing paradigms in the field of bacterial gene regulation. The Wadsworth Center provides an outstanding environment for these studies because of its excellent research into bacteria and gene regulation, and a strong network of core facilities. The work proposed here will transform our understanding of bacterial gene expression and has major implications for the study of bacterial physiology and pathogenesis. Public Health Relevance: RNA has recently been recognized as a key regulator of gene expression. We have identified over 1,000 novel RNAs in the model bacterium, Escherichia coli. We will determine the impact of these RNAs on gene expression.

National Institute of Health (NIH)
Office of The Director, National Institutes of Health (OD)
NIH Director’s New Innovator Awards (DP2)
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Special Emphasis Panel (ZGM1-NDIA-O (01))
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Basavappa, Ravi
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Wadsworth Center
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Smith, Carol; Stringer, Anne M; Mao, Chunhong et al. (2016) Mapping the Regulatory Network for Salmonella enterica Serovar Typhimurium Invasion. MBio 7:
Kasper, Stephen H; Bonocora, Richard P; Wade, Joseph T et al. (2016) Chemical Inhibition of Kynureninase Reduces Pseudomonas aeruginosa Quorum Sensing and Virulence Factor Expression. ACS Chem Biol 11:1106-17
Singh, Navjot; Bubunenko, Mikhail; Smith, Carol et al. (2016) SuhB Associates with Nus Factors To Facilitate 30S Ribosome Biogenesis in Escherichia coli. MBio 7:e00114
Bonocora, Richard P; Wade, Joseph T (2015) ChIP-seq for genome-scale analysis of bacterial DNA-binding proteins. Methods Mol Biol 1276:327-40
Wang, Jing; Rennie, William; Liu, Chaochun et al. (2015) Identification of bacterial sRNA regulatory targets using ribosome profiling. Nucleic Acids Res 43:10308-20
Bonocora, Richard P; Smith, Carol; Lapierre, Pascal et al. (2015) Genome-Scale Mapping of Escherichia coli σ54 Reveals Widespread, Conserved Intragenic Binding. PLoS Genet 11:e1005552
Singh, Navjot; Wade, Joseph T (2014) Identification of regulatory RNA in bacterial genomes by genome-scale mapping of transcription start sites. Methods Mol Biol 1103:1-10
Wade, Joseph T; Grainger, David C (2014) Pervasive transcription: illuminating the dark matter of bacterial transcriptomes. Nat Rev Microbiol 12:647-53
Fitzgerald, Devon M; Bonocora, Richard P; Wade, Joseph T (2014) Comprehensive mapping of the Escherichia coli flagellar regulatory network. PLoS Genet 10:e1004649
Singh, Shivani S; Singh, Navjot; Bonocora, Richard P et al. (2014) Widespread suppression of intragenic transcription initiation by H-NS. Genes Dev 28:214-9

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