Sequences in the -35 region of Escherichia coli rpoS-dependent genes promote transcription by E sigma(s)

sigma(S) is an alternate sigma factor which functions with RNA polymerase to activate transcription of genes that are involved in a number of stress responses, including stationary-phase survival and osmoprotection. The similarity of the sigma(S) protein to sigma(D) (Escherichia coli's major sigma factor) in the regions thought to recognize and bind promoter sequences suggests that sigma(S) - and sigma(D)-associated RNA polymerases recognize promoter DNA in a similar manner. However, no promoter recognition sequence for sigma(S) holoenzyme (E sigma(S)) has been identified. An apparent conservation of cytosine nucleotides was noted in the -35 region of several sigma(S)-dependent promoters. Site directed mutagenesis and reporter gene fusions were used to investigate the importance of the -35 cytosine nucleotides for sigma(S)-dependent transcription. Substitution of cytosine nucleotides for thymidine at the -35 site of the sigma(D)-dependent proU promoter effectively abolished transcription by E sigma(D) but allowed E sigma(S) to direct transcription from the mutant promoter. Inclusion of the sigma(D) consensus -10 hexamer strengthened transcription by E sigma(S), demonstrating that both E sigma(D) and E sigma(S) can recognize the same -10 sequences. Conversely, replacement of -35 site cytosine nucleotides with thymidine in the sigma(S)-dependent osmY promoter reduced transcription by E sigma(S) and increased transcription by E sigma(D). Our data suggest that DNA sequences in the -35 region function as part of a discriminator mechanism to shift transcription between E sigma(D) and E sigma(S).