Autoregulation-deficient mutant of the plasmid R6K-encoded pi protein distinguishes between palindromic and nonpalindromic binding sites.
The autogenously regulated gene pir of Escherichia coli plasmid R6K encodes the replication protein pi. This protein binds to two sites in the operator region of the pir gene: a 22-base pair nonpalindromic sequence and a pair of palindromic 9-base pair sequences. These pi-binding sites are similar, suggesting that pi uses a single DNA-binding domain in recognizing them. We devised a plasmid system permitting isolation of mutants of the pi protein which are altered in autoregulation. A Ser87 to Asn substitution in one such mutant, designated pi 87, reduces the protein's ability to repress the pir gene promoter in vivo. DNase I protection and gel retardation assays were carried out with highly purified pi 87 protein. In these studies pi 87 exhibited altered binding to the palindromic but not to the nonpalindromic part of the operator of the pir gene. Chemical cross-linking and gel filtration analyses have shown that the dimerization properties of wild type pi and pi 87 proteins are similar in solution. We propose that the interaction of pi protein with the palindromic part of the pir operator is essential for autoregulation; we also propose that there is a fundamental difference in the mechanisms of pi protein recognition of palindromic and nonpalindromic sequences.
York
D
D
Department of Bacteriology, University of Wisconsin, Madison 53706.
Filutowicz
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004265:DNA Helicases; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D009154:Mutation; D009875:Operator Regions, Genetic; D010448:Peptide Initiation Factors / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D010448:Peptide Initiation Factors; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases