Indirect read-out of the promoter DNA by RNA polymerase in the closed complex

Abstract

Transcription is initiated when RNA polymerase recognizes the duplex promoter DNA in the closed complex. Due to its transient nature, the closed complex has not been well characterized. How the initial promoter recognition occurs may offer important clues to regulation of transcription initiation. In this article, we have carried out single-base pair substitution experiments on two Escherichia coli promoters belonging to two different classes, the −35 and the extended −10, under conditions which stabilize the closed complex. Single-base pair substitution experiments indicate modest base-specific effects on the stability of the closed complex of both promoters. Mutations of base pairs in the −10 region affect the closed complexes of two promoters differently, suggesting different modes of interaction of the RNA polymerase and the promoter in the two closed complexes. Two residues on σ70 which have been suggested to play important role in promoter recognition, Q437 and R436, were mutated and found to have different effects on the closed-complex stability. DNA circular dichroism (CD) and FRET suggest that the promoter DNA in the closed complex is distorted. Modeling suggests two different orientations of the recognition helix of the RNA polymerase in the closed complex. We propose that the RNA polymerase recognizes the sequence dependent conformation of the promoter DNA in the closed complex.