Sequence specificity for DNA binding by Escherichia coli SoxS and Rob proteins.
SoxS is a transcriptional activator of oxidative stress genes in Escherichia coli. SoxS in vitro binds the promoters of soxRS-regulated genes such as micF, zwf, nfo and sodA, forms multiple protein-DNA complexes, and recruits RNA polymerase to the promoters. E. coli Rob protein, with an N-terminus 55% identical to SoxS, was initially identified by its binding to the oriC replication origin, but Rob in vitro binds some of the same promoters as SoxS and in vivo activates some SoxS-regulated genes. In this work we show that the multiple complexes with SoxS arise from the presence at least two independent binding sites in each of the ++offcF and zwf promoters. SoxS and Rob each form only a single complex with a 20 bp DNA oligonucleotide corresponding to the region immediately upstream of the -35 element of the micF promoter. Methylation interference identified several conserved purine residues required for binding to micF and five other SoxS-binding sites. Together with binding studies using mutated ollgonucleotides and published DNase I footprinting data, this information was used to form a consensus for SoxS sequence specificity: AN2GCAYN7CWA (where N is any base, Y is a pyrimidine, and W is A or T). The sequence requirements for Rob binding differed somewhat from those of SoxS. Using the SoxS-binding consensus, several genes potentially regulated by soxRS were identified in an E. coli genomic database; some of these genes have functions that might contribute to cellular resistance to oxidative stress.
Li
Z
Z
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Demple
B
B
D001426:Bacterial Proteins / Q000378:metabolism*; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D017124:Conserved Sequence; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; C080267:Rob protein, E coli; D015534:Trans-Activators; D014157:Transcription Factors; C070725:SoxS protein, E coli