title: 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.
abstract: Specific promoter recognition by bacterial RNA polymerase is mediated by σ subunits, which assemble with RNA polymerase core enzyme (E) during transcription initiation. However, σ(70) (the housekeeping σ subunit) and σ(S) (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 σ(70) (Eσ(70)) or with σ(S) (Eσ(S)) using the whole Escherichia coli genome as DNA template. We found that Eσ(70), in the absence of any additional transcription factor, preferentially transcribes genes associated with fast growth (e.g. ribosomal operons). In contrast, Eσ(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 Eσ(S).
authors.affiliation: Department of Biomolecular Sciences and Biotechnology, Università degli Studi di Milano, Milan, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Maciag
authors.forename: Clelia
authors.initials: C
authors.lastname: Peano
authors.forename: Alessandro
authors.initials: A
authors.lastname: Pietrelli
authors.forename: Thomas
authors.initials: T
authors.lastname: Egli
authors.forename: Gianluca
authors.initials: G
authors.lastname: De Bellis
authors.forename: Paolo
authors.initials: P
authors.lastname: Landini
mesh_terms: D001483:Base Sequence; D017124:Conserved Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D020869:Gene Expression Profiling; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D021122:Protein Subunits / Q000378:metabolism; D018085:Regulon; D012808:Sigma Factor / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D021122:Protein Subunits; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases