6S RNA binding to Esigma(70) requires a positively charged surface of sigma(70) region 4.2.

6S RNA is a small, non-coding RNA that interacts with sigma(70)-RNA polymerase and downregulates transcription at many promoters during stationary phase. When bound to sigma(70)-RNA polymerase, 6S RNA is engaged in the active site of sigma(70)-RNA polymerase in a manner similar enough to promoter DNA that the RNA can serve as a template for RNA synthesis. It has been proposed that 6S RNA mimics the conformation of DNA during transcription initiation, suggesting contacts between RNA polymerase and 6S RNA or DNA may be similar. Here we demonstrate that region 4.2 of sigma(70) is critical for the interaction between 6S RNA and RNA polymerase. We define an expanded binding surface that encompasses positively charged residues throughout the recognition helix of the helix-turn-helix motif in region 4.2, in contrast to DNA binding that is largely focused on the N-terminal region of this helix. Furthermore, negatively charged residues in region 4.2 weaken binding to 6S RNA but do not similarly affect DNA binding. We propose that the binding sites for promoter DNA and 6S RNA on region 4.2 of sigma(70) are overlapping but distinct, raising interesting possibilities for how core promoter elements contribute to defining promoters that are sensitive to 6S RNA regulation.