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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057755-30
Application #
8300931
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Sledjeski, Darren D
Project Start
1983-01-01
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
30
Fiscal Year
2012
Total Cost
$472,112
Indirect Cost
$161,928
Name
University of California San Francisco
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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
Rhodius, Virgil A; Segall-Shapiro, Thomas H; Sharon, Brian D et al. (2013) Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, 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
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
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 (2010) Predicting strength and function for promoters of the Escherichia coli alternative sigma factor, sigmaE. Proc Natl Acad Sci U S A 107:2854-9
Mutalik, Vivek K; Nonaka, Gen; Ades, Sarah E et al. (2009) Promoter strength properties of the complete sigma E regulon of Escherichia coli and Salmonella enterica. J Bacteriol 191:7279-87
Koo, Byoung-Mo; Rhodius, Virgil A; Nonaka, Gen et al. (2009) Reduced capacity of alternative sigmas to melt promoters ensures stringent promoter recognition. Genes Dev 23:2426-36
Rhodius, Virgil A; Wade, Joseph T (2009) Technical considerations in using DNA microarrays to define regulons. Methods 47:63-72

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