The broad, long range goal of this proposal is to learn how genes are controlled by remotely bound activator proteins. This is a problem of great significance since the transcription of most genes in human cells is controlled in this manner. These genes include oncogenes and those controlling the production of other proteins whose functions are critical for the health of the cells and the organism. This process is of such complexity and interest that an enormous scientific effort is currently being applied to its solution. However, the recent discovery of an analogous process in bacterial cells provides entry into studying the problem in a much simpler and more accessible system. A rare bacterial protein, sigma 54, resembles closely analogous proteins in mammalian cells and is required to mediate the control from distant DNA sites in E. Coli. The mechanism of action of sigma 54 will be determined in this proposal. the entire length of the protein will be covered by mutations and the mutant proteins expressed from plasmids in cells lacking the wild-type protein. In vivo probing, genetic screens, and selected in vitro experiments will be applied to learn the roles of each part of the protein in mediating this long range control. The way in which these domains interact with each other and with the DNA and the other components of the transcription apparatus will be determined. New methods of in vivo probing will be developed in order to speed and extend this work. It is expected that the analysis of this simple system will precede analysis of more complex systems and thus help guide the tremendous and important ongoing effort to learn the role of oncogene control proteins and other analogous factors in mammalian cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035754-08
Application #
3288917
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1985-12-01
Project End
1994-11-30
Budget Start
1992-12-01
Budget End
1993-11-30
Support Year
8
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Arts and Sciences
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Gralla, Jay D; Huo, Yi-Xin (2008) Remodeling and activation of Escherichia coli RNA polymerase by osmolytes. Biochemistry 47:13189-96
Huo, Yi-Xin; Rosenthal, Adam Z; Gralla, Jay D (2008) General stress response signalling: unwrapping transcription complexes by DNA relaxation via the sigma38 C-terminal domain. Mol Microbiol 70:369-78
Rosenthal, Adam Z; Kim, Youngbae; Gralla, Jay D (2008) Poising of Escherichia coli RNA polymerase and its release from the sigma 38 C-terminal tail for osmY transcription. J Mol Biol 376:938-49
Rosenthal, Adam Z; Kim, Youngbae; Gralla, Jay D (2008) Regulation of transcription by acetate in Escherichia coli: in vivo and in vitro comparisons. Mol Microbiol 68:907-17
Gralla, Jay D; Vargas, David R (2006) Potassium glutamate as a transcriptional inhibitor during bacterial osmoregulation. EMBO J 25:1515-21
Kim, Youngbae; Lew, Chih M; Gralla, Jay D (2006) Escherichia coli pfs transcription: regulation and proposed roles in autoinducer-2 synthesis and purine excretion. J Bacteriol 188:7457-63
Gralla, Jay D (2005) Escherichia coli ribosomal RNA transcription: regulatory roles for ppGpp, NTPs, architectural proteins and a polymerase-binding protein. Mol Microbiol 55:973-7
Lew, Chih M; Gralla, Jay D (2004) Nucleotide-dependent isomerization of Escherichia coli RNA polymerase. Biochemistry 43:12660-6
Lee, Shun Jin; Gralla, Jay D (2004) Osmo-regulation of bacterial transcription via poised RNA polymerase. Mol Cell 14:153-62
Lee, Shun Jin; Gralla, Jay D (2003) Open complex formation in vitro by sigma38 (rpoS) RNA polymerase: roles for region 2 amino acids. J Mol Biol 329:941-8

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