A Hydrophobic Patch on the Flap-tip Helix of E. coli RNA Polymerase Mediates σ70 Region 4 Function

The Escherichia coli RNA polymerase β subunit contains a flexible flap domain that interacts with region 4 of σ70 to position it for recognition of the −35 element of promoters. We report that this function depends on a hydrophobic patch on one face of the short stretch of α helix located at the tip of the flap domain, called the flap-tip helix. Disruption of the hydrophobic patch by the substitution of hydrophilic or charged amino acids resulted in a loss of the interaction between the flap and σ region 4, as determined by protease sensitivity assays, and impaired transcription from −35-dependent promoters. We suggest that contact of the flap-tip helix hydrophobic patch to the σ region 4 hydrophobic core is essential for stable interaction of the flap-tip helix with region 4. This contact allowed region 4.2 recognition of the −35 promoter element and appeared to stabilize region 4 interaction with the β′ Zn2+ binding domain. Our studies failed to detect any role for σ region 1.1 in establishing or maintaining the flap–σ region 4 interaction, consistent with recent reports placing σ region 1.1 in the downstream DNA channel.