In vitro transcription profiling of the Sigma(S) subunit of bacterial RNA polymerase: re-definition of the Sigma(S) regulon and identification of Sigma(S)-specific promoter sequence elements

Specific promoter recognition by bacterial RNA polymerase is mediated by Sigma subunits, which assemble with RNA polymerase core enzyme (E) during transcription initiation. However, Sigma(70) (the housekeeping Sigma subunit) and Sigma(S) (an alternative Sigma subunit mostly active during slow growth) recognize almost identical promoter sequences, thus raising the question of how promoter selectivity is achieved in the bacterial cell. To identify novel sequence determinants for selective promoter recognition, we performed run-off/microarray (ROMA) experiments with RNA polymerase saturated either with Sigma(70) (E Sigma(70)) or with Sigma(S) (E Sigma(S)) using the whole Escherichia coli genome as DNA template. We found that E Sigma(70), in the absence of any additional transcription factor, preferentially transcribes genes associated with fast growth (e.g. ribosomal operons). In contrast, E Sigma(S) efficiently transcribes genes involved in stress responses, secondary metabolism as well as RNAs from intergenic regions with yet-unknown function. Promoter sequence comparison suggests that, in addition to different conservation of the -35 sequence and of the UP element, selective promoter recognition by either form of RNA polymerase can be affected by the A/T content in the -10/+1 region. Indeed, site-directed mutagenesis experiments confirmed that an A/T bias in the -10/+1 region could improve promoter recognition by E Sigma(S).