We study how the cell controls information flow from genes into proteins. In bacteria, primary control is at the transcriptional level, orchestrated by multiple sigmas (?s). Housekeeping ?s direct transcription from 1000's of promoters;alternative ?s direct transcription from specific genes to cope with environmental change. Our goal is to understand the interplay between ?s and RNA polymerase (RNAP), and the network properties that govern RNAP output. We bring to this work a long history of providing insight into the roles of ?s and a new genomic technology. In the current granting period, we will investigate three fundamental and unsolved issues: (1) The alternative """"""""stress"""""""" ?s increase transcription from a limited gene set necessary to cope with the stress;we will determine features of alternative ?s that result in stringent promoter selection. (2) We have been developing new paradigms to predict alternative ?s. Using combinatorial libraries and high throughput approaches, we will critically test the ability of current algorithms to predict promoters of the ?E stress ?, and develop new algorithms that function better. (3) Transcription initiation has been studied largely in isolation. Using our new genomic methodology for high throughput, quantitative determination of genetic interactions, we will determine how RNAP is integrated into the broader workings of the bacterial cell. These benchmark studies will be made available immediately in a public database, thereby moving the entire field forward by identifying new proteins and functional relationships in transcription and new relationships among cellular processes. We perform these studies primarily in E. coli, where a wealth of genetic, biochemical and physiological data facilitate analysis, but our results are directly applicable to all bacteria, including pathogens and environmentally beneficial microbes. Moreover, the questions we are asking and the methods we are developing allow us to directly extend our studies to other bacteria that may be difficult or even impossible to study experimentally. Finally, our studies have important direct applications. The family of circuits we are dissecting may provide building blocks for synthetic circuits with diverse output properties. Additionally, the observation that many RNAP mutants have enhanced growth capabilities under particular conditions or increased resistance to environmental stress indicates that RNAP is a hotspot for adaptive evolution;understanding the mechanism behind these effects could be of use in drug discovery efforts, metabolic engineering, biofuel production and bioremediation.

Public Health Relevance

We study the molecular machineries and circuitries that govern gene expression in bacteria and enable them to proliferate under diverse and challenging environments. This information allows us to manipulate and control bacteria, including pathogens and environmentally beneficial microorganisms. Additionally, our work is of direct relevance to drug discovery, metabolic engineering, biofuel production and bioremediation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Tompkins, Laurie
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University of California San Francisco
Schools of Medicine
San Francisco
United States
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Burkhardt, David H; Rouskin, Silvi; Zhang, Yan et al. (2017) Operon mRNAs are organized into ORF-centric structures that predict translation efficiency. Elife 6:
Shiver, Anthony L; Osadnik, Hendrik; Kritikos, George et al. (2016) A Chemical-Genomic Screen of Neglected Antibiotics Reveals Illicit Transport of Kasugamycin and Blasticidin S. PLoS Genet 12:e1006124
Gross, Carol A; Gründling, Angelika (2015) Editorial overview: Cell regulation: when you think you know it all, there is another layer to be discovered. Curr Opin Microbiol 24:v-vii
Parshin, Andrey; Shiver, Anthony L; Lee, Jookyung et al. (2015) DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1. Proc Natl Acad Sci U S A 112:E6862-71
Gray, Andrew N; Koo, Byoung-Mo; Shiver, Anthony L et al. (2015) High-throughput bacterial functional genomics in the sequencing era. Curr Opin Microbiol 27:86-95
Darst, Seth A; Feklistov, Andrey; Gross, Carol A (2014) Promoter melting by an alternative ?, one base at a time. Nat Struct Mol Biol 21:350-1
Rhodius, Virgil A; Segall-Shapiro, Thomas H; Sharon, Brian D et al. (2013) Design of orthogonal genetic switches based on a crosstalk map of ?s, anti-?s, and promoters. Mol Syst Biol 9:702
McCusker, Kevin P; Medzihradszky, Katalin F; Shiver, Anthony L et al. (2012) Covalent intermediate in the catalytic mechanism of the radical S-adenosyl-L-methionine methyl synthase RlmN trapped by mutagenesis. J Am Chem Soc 134:18074-81
Dufour, Yann S; Imam, Saheed; Koo, Byoung-Mo et al. (2012) Convergence of the transcriptional responses to heat shock and singlet oxygen stresses. PLoS Genet 8:e1002929
Rhodius, Virgil A; Mutalik, Vivek K; Gross, Carol A (2012) Predicting the strength of UP-elements and full-length E. coli ýýE promoters. Nucleic Acids Res 40:2907-24

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