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

Maciąg, Anna, Peano, Clelia, Pietrelli, Alessandro, Egli, Thomas, De Bellis, Gianluca, Landini, Paolo
Nucleic acids research 2011 v.39 no.13 pp. 5338-5355
DNA-directed RNA polymerase, Escherichia coli, RNA, bacteria, intergenic DNA, metabolism, operon, promoter regions, regulon, sequence analysis, site-directed mutagenesis, stress response, transcription (genetics), transcription factors
Specific promoter recognition by bacterial RNA polymerase is mediated by σ subunits, which assemble with RNA polymerase core enzyme (E) during transcription initiation. However, σ⁷⁰ (the housekeeping σ subunit) and σS (an alternative σ 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 σ⁷⁰ (Eσ⁷⁰) or with σS (EσS) using the whole Escherichia coli genome as DNA template. We found that Eσ⁷⁰, in the absence of any additional transcription factor, preferentially transcribes genes associated with fast growth (e.g. ribosomal operons). In contrast, EσS 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 EσS.