E. coli RNA polymerase binds promoter DNA to a channel on its surface and is able to melt about 12 base pairs of the promoter DNA to form a strand separated or """"""""open"""""""" complex. On the pathway to formation of this complex, several distinct intermediate complexes have been recognized. The transition between two of them likely involves a major conformational change in the RNA polymerase, triggering the nucleation of strand separation in the - 10 region, from where basepair disruption propagates in downstream direction. Three additional regions of sequence-specific interaction between E. coli RNA polymerase and promoters have been recognized: a TG dinucleotide just upstream of the -10 element, the -35 region and the UP element at -40 through -60. We propose to investigate several aspects of the interaction of RNA polymerase with nucleic acids, and the role of sigma factor in the process. Using several different approaches we will better define the role of the TG, -35 and UP sequences in facilitating open complex formation. We will continue our studies on the interaction of RNA polymerase with single stranded (ss) DNA, and characterize the conformational change in RNA polymerase that accompanies ss DNA binding. We will also initiate the characterization of the thermodynamics of the interaction between ss DNA and the RNA polymerase of a thermophile (optimally active at 55xC). The group of transcription initiation factors known as sigma factors is responsible for the interaction with promoter DNA at the -10 and -35 regions, the TG sequence and ss DNA in the open complex. We will determine he role of highly conserved residues of sigma factor in open complex formation by introduction of alanine substitutions in a region spanning conserved regions 2.2, 2.3 and 2.4. Selected substitutions will be combined to generate sigma factors severely defective in the DNA melting step. These will be useful in probing the process of nucleation of DNA strand separation. In addition we will study RNA polymerase containing sigma32, which differs from sigma70 in conserved region 2.3, and also in the promoter sequence it recognizes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031808-18
Application #
6385480
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Tompkins, Laurie
Project Start
1983-04-01
Project End
2003-08-31
Budget Start
2001-09-01
Budget End
2002-08-31
Support Year
18
Fiscal Year
2001
Total Cost
$311,057
Indirect Cost
Name
Case Western Reserve University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
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
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
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
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

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