Abstract

The process of copying information contained in the DNA of an organism into RNA by RNA polymerases is a crucial step in the biological expression of this information. Consequently, this is an important junction at which control of gene expression can be exerted. I propose to study a well characterized system, the interaction of Escherichia coli RNA polymerase with its specific functional sites on DNA (promoters), and the control of the process by the cI protein of Phage Lamda. I will focus on two regions of DNA with which the RNA polymerase makes contact when bound to a promoter, and the spacer DNA separating them. The interactions of RNA polymerase with each of the two regions will be studied separately. The effect of altering the relative rotational orientation of the two DNA regions on promoter functioning will be explored by modifications of the sequence of the spacer DNA rather than changing its length. These modifications will be introduced using newly developed rapid methods for chemical DNA synthesis, which allows for great versatility in manipulating DNA sequences. Cloning of the synthesized DNA will aid its manipulation and enable the testing of the modified promoters in vivo. A variety of biochemical and physical techniques will be used to determine the binding affinity of RNA polymerase for the constructed DNA substrates, the extend of distortion of spacer DNA by bound RNA polymerase, and the in vitro and in vivo functioning of the altered promoters. Many of the same promoter variants will be used to characterize in detail the mechanism of action of the weak phage promoters by the phage encoded cI protein. The intended research should significantly further our understanding of those determinants on the DNA of weak promoters which allow their activation by controlling proteins, as well as of those determinants which necessitate such an activation. These studies will provide the foundation to explore the regulation of RNA polymerase III catalyzed transcription in eukaryotic organisms.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031808-03
Application #
3280128
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1983-04-01
Project End
1986-06-30
Budget Start
1985-04-01
Budget End
1986-06-30
Support Year
3
Fiscal Year
1985
Total Cost
Indirect Cost

  Institution

Name
Case Western Reserve University
Department
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106

  Publications

Saecker, Ruth M; Record Jr, M Thomas; Dehaseth, Pieter L (2011) Mechanism of bacterial transcription initiation: RNA polymerase - promoter binding, isomerization to initiation-competent open complexes, and initiation of RNA synthesis. J Mol Biol 412:754-71
Schroeder, Lisa A; Gries, Theodore J; Saecker, Ruth M et al. (2009) Evidence for a tyrosine-adenine stacking interaction and for a short-lived open intermediate subsequent to initial binding of Escherichia coli RNA polymerase to promoter DNA. J Mol Biol 385:339-49
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
Schroeder, Lisa A; Karpen, Mary E; deHaseth, Pieter L (2008) Threonine 429 of Escherichia coli sigma 70 is a key participant in promoter DNA melting by RNA polymerase. J Mol Biol 376:153-65
Cook, Victoria M; Dehaseth, Pieter L (2007) Strand opening-deficient Escherichia coli RNA polymerase facilitates investigation of closed complexes with promoter DNA: effects of DNA sequence and temperature. J Biol Chem 282:21319-26
Schroeder, Lisa A; Choi, Ae-Jin; DeHaseth, Pieter L (2007) The -11A of promoter DNA and two conserved amino acids in the melting region of sigma70 both directly affect the rate limiting step in formation of the stable RNA polymerase-promoter complex, but they do not necessarily interact. Nucleic Acids Res 35:4141-53
Kourennaia, Olga V; Dehaseth, Pieter L (2007) Substitution of a highly conserved histidine in the Escherichia coli heat shock transcription factor, sigma32, affects promoter utilization in vitro and leads to overexpression of the biofilm-associated flu protein in vivo. J Bacteriol 189:8430-6
Schroeder, Lisa A; deHaseth, Pieter L (2005) Mechanistic differences in promoter DNA melting by Thermus aquaticus and Escherichia coli RNA polymerases. J Biol Chem 280:17422-9
Kourennaia, Olga V; Tsujikawa, Laura; Dehaseth, Pieter L (2005) Mutational analysis of Escherichia coli heat shock transcription factor sigma 32 reveals similarities with sigma 70 in recognition of the -35 promoter element and differences in promoter DNA melting and -10 recognition. J Bacteriol 187:6762-9
Sun, Li; Dove, Simon L; Panaghie, Gianina et al. (2004) An RNA polymerase mutant deficient in DNA melting facilitates study of activation mechanism: application to an artificial activator of transcription. J Mol Biol 343:1171-82

Showing the most recent 10 out of 45 publications