A hydrophobic patch on the flap-tip helix of E-coli RNA polymerase mediates σ70 region 4 function

The Escherichia coli RNA polymerase beta subunit contains a flexible flap domain that interacts with region 4 of sigma(70) 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 alpha 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 sigma 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 a 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 beta' Zn2+ binding domain. Our studies failed to detect any role for a region 1.1 in establishing or maintaining the flap-sigma region 4 interaction, consistent with recent reports placing sigma region 1.1 in the downstream DNA channel. (C) 2004 Elsevier Ltd. All rights reserved.