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

Abstract

Bacterial RNA polymerase and a “sigma” transcription factor form an initiation-competent “open” complex at a promoter by disruption of about 14 base pairs. Strand separation is likely initiated at the highly conserved − 11 A–T base pair. Amino acids in conserved region 2.3 of the main Escherichia coli sigma factor, σ 70, are involved in this process, but their roles are unclear. To monitor the fates of particular bases upon addition of RNA polymerase, promoters bearing single substitutions of the fluorescent A-analog 2-aminopurine (2-AP) at − 11 and two other positions in promoter DNA were examined. Evidence was obtained for an open intermediate on the pathway to open complex formation, in which these 2-APs are no longer stacked onto their neighboring bases. The tyrosine at residue 430 in region 2.3 of σ 70 was shown to be involved in quenching the fluorescence of a 2-AP substituted at − 11, presumably through a stacking interaction. These data refine the structural model for open complex formation and reveal a novel interaction involved in DNA melting by RNA polymerase.