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Identification of promoter sequence elements involved in specific recognition by the σS subunit of bacterial RNA polymerase.

ABSTRACT: Promoter recognition by bacterial RNA polymerase is mediated by σ subunits, which assemble transiently to RNA polymerase core enzyme (E) during transcription initiation. σ subunits drive transcription of specific sets of genes by allowing RNA polymerase to interact with different promoter sequences. However, σ70, the housekeeping σ subunit, and σS, an alternative σ subunit mainly active during slow growth and in response to cellular stresses, appear to recognize almost identical promoter sequences, raising the question of how promoter selectivity is achieved in the bacterial cell. To identify sequence determinants for selective promoter recognition, we performed a run-off/microarray experiment (ROMA): in vitro transcription experiments were carried out with RNA polymerase saturated either with σ70 (Eσ70) or with σS (EσS) using the whole Escherichia coli genome as DNA template, and transcript levels were determined by microarray analysis. We found that several genes associated with bacterial growth (e.g., ribosomal operons) were transcribed more efficiently by Eσ70. In contrast, EσS transcribed preferentially genes involved in stress responses, secondary metabolism, as well as regulatory RNAs and intergenic regions with yet unknown function. Genes preferentially recognized in vitro by EσS showed reduced expression in EσS -deficient mutant strain of E. coli. Sequence comparison of Eσ70- versus EσS –dependent promoters confirms that the presence of a -35 sequence and the relative location of UP elements affect promoter interaction with either form of RNA polymerase, and suggests that a G/C bias in the -2/+1 nucleotides would favour efficient promoter recognition by Eσ70.

ORGANISM(S): Escherichia coli Escherichia coli str. K-12 substr. MG1655

PROVIDER: GSE22207 | GEO | 2011/05/31

SECONDARY ACCESSION(S): PRJNA128873

REPOSITORIES: GEO

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Project description:Background: In prokaryotes, sigma factors are essential for the targeting of the transcription machinery to promoters. Various sigma factors have been described that recognize and bind to specific DNA sequence motifs in promoter sequences. The canonical sigma factor σ70 is commonly involved in transcription of the cell's general housekeeping genes, which is mediated by the conserved σ70 promoter sequence motifs. This study detects and predicts the general σ70-promoter sequences in Lactobacillus plantarum WCFS1 using genome-wide analysis. The accuracy of the transcriptionally-active part of this promoter prediction was subsequently evaluated by correlating locations of predicted promoters with experimentally detected transcription starts sites (TSSs) using high-resolution tiling array transcriptome datasets. Results: To identify σ70-related promoter sequences, we performed a genome-wide sequence motif scan of the L. plantarum WCFS1 genome focusing on the regions upstream of protein-encoding genes. We obtained several highly conserved motifs including those resembling the conserved σ70-promoter consensus. Position weight matrices-based models of the recovered σ70-promoter sequence motif were employed to identify 4746 motifs with significant similarity (p-value < 10-4) to the model-motif in the L. plantarum genome. Genome-wide transcription start site information deduced from whole genome tiling-array transcriptome datasets revealed 936 TSSs that were employed to validate the transcriptionally active fraction of these predicted promoters. In total, 578 predicted promoters were found in proximity (≤ 100 nucleotides) of the identified TSSs, showing a highly significant co-occurrence of predicted promoter and measured TSS (p-value < 10-23). An additional 224 predicted promoters was validated when the significant similarity to the model-motif was applied less strictly (10-4 ≤ p-value < 10-3). Conclusions: High-resolution tiling arrays provide a suitable source for TSS detection at a genome-wide level, and allow experimental verification of in silico-predicted promoter sequence motifs.

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Identification of promoter sequence elements involved in specific recognition by the σS subunit of bacterial RNA polymerase.

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