Nucleic Acids Research Advance Access published online on June 12, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm431
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Nucleic Acid Enzymes |
The –11A of promoter DNA and two conserved amino acids in the melting region of 
70 both directly affect the rate limiting step in formation of the stable RNA polymerase-promoter complex, but they do not necessarily interact
1The Center for RNA Molecular Biology and 2The Department of Biochemistry, Case Western Reserve University, Cleveland OH, USA
*To whom correspondence should be addressed. Tel: 216 368 5045; Fax: 216 368 2010; Email: las30{at}case.edu
Received February 2, 2007. Revised April 20, 2007. Accepted May 14, 2007.
Formation of the stable, strand separated, ‘open’ complex between RNA polymerase and a promoter involves DNA melting of approximately 14 base pairs. The likely nucleation site is the highly conserved –11A base in the non-template strand of the –10 promoter region. Amino acid residues Y430 and W433 on the 
70 subunit of the RNA polymerase participate in the strand separation. The roles of –11A and of the Y430 and W433 were addressed by employing synthetic consensus promoters containing base analog and other substitutions at –11 in the non-template strand, and 70 variants bearing amino acid substitutions at positions 430 and 433. Substitutions for –11A and for Y430 and W433 in 70 have small or no effects on formation of the initial RNA polymerase-promoter complex, but exert their effects on subsequent steps on the way to formation of the open complex. As substitutions for Y430 and W433 also affect open complex formation on promoter DNA lacking the –11A base, it is concluded that these amino acid residues have other (or additional) roles, not involving the –11A. The effects of the substitutions at –11A of the promoter and Y430 and W433 of 70 are cumulative.
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