Threonine 429 of E. coli σ70 is a key participant in promoter DNA melting by RNA polymerase

• Published in: Journal of molecular biology 2007-11, Vol.376 (1), p.153-165
• Main Authors: Schroeder, Lisa A., Karpen, Mary E., deHaseth, Pieter L.
• Format: Article
• Language: English
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2007.11.070

Initiation of transcription is an important target for regulation of gene expression. In bacteria, formation of a transcription-competent complex between RNA polymerase and a promoter involves DNA strand separation over a stretch of about 14 base pairs. Aromatic and basic residues in conserved region 2.3 of E. coli σ 70 had been found to participate in this process, but it is still unclear which amino acid residues initiate it. Here we report an essential role for the threonine (T) at position 429 of σ 70 : its substitution by alanine (T429A) results in the largest decrease in open complex formation yet observed for any single substitution in region 2.3. Promoter recognition itself is not affected by the T429A substitution, thus providing evidence for a role of T429 in the strand separation step. Our data are consistent with a model where the T429 would act as a competitor for the hydrogen bonding stabilizing the highly conserved -11 A-T base pair of the promoter DNA, thus facilitating initiation of strand separation at this particular position in the -10 region. This model suggests an active role for RNA polymerase in disrupting the -11 base pair, rather than just capturing the -11A subsequent to spontaneous unpairing.