Tandem binding of six OmpR proteins to the ompF upstream regulatory sequence of Escherichia coli.
OmpR is a transcription factor in Escherichia coli whose function is modulated by phosphorylation in the presence of phosphorylated EnvZ, a transmembrane protein histidine kinase involved in osmosensing. Using a protein S-OmpR hybrid protein, we demonstrated that six OmpR molecules bind tandemly to the -100 to -39 sequence of ompF. This sequence consists of three 20-base pair units: F1, F2, and F3, each of which is bound by two OmpR proteins. Polymerase chain reaction selection of nine randomized base pairs within the F1 sequence revealed highly conserved C residues spaced 10 base pairs apart. Further mutational analysis of conserved bases indicated that two OmpR molecules bind tandemly to two direct repeats. Mobility shift assays showed that cooperative interactions play a role in enhancing binding of OmpR to lower affinity F2 and F3 sites. Activation and repression of ompF expression are thus regulated by a total of eight OmpR molecules, including two molecules that bind to a distal site (-380 to -361).
Harlocker
S L
SL
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Bergstrom
L
L
Inouye
M
M
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016384:Consensus Sequence; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005809:Genes, Regulator; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors