title: The helix-turn-helix motif of the coliphage 186 immunity repressor binds to two distinct recognition sequences.
abstract: The CI protein of coliphage 186 is responsible for maintaining the stable lysogenic state. To do this CI must recognize two distinct DNA sequences, termed A type sites and B type sites. Here we investigate whether CI contains two separate DNA binding motifs or whether CI has one motif that recognizes two different operator sequences. Sequence alignment with 186-like repressors predicts an N-terminal helix-turn-helix (HTH) motif, albeit with poor homology to a large master set of such motifs. The domain structure of CI was investigated by linker insertion mutagenesis and limited proteolysis. CI consists of an N-terminal domain, which weakly dimerizes and binds both A and B type sequences, and a C-terminal domain, which associates to octamers but is unable to bind DNA. A fusion protein consisting of the 186 N-terminal domain and the phage lambda oligomerization domain binds A and B type sequences more efficiently than the isolated 186 CI N-terminal domain, hence the 186 C-terminal domain likely mediates oligomerization and cooperativity. Site-directed mutation of the putative 186 HTH motif eliminates binding to both A and B type sites, supporting the idea that binding to the two distinct DNA sequences is mediated by a variant HTH motif.
authors.affiliation: Department of Molecular Biosciences, University of Adelaide, Adelaide, Australia 5005. keith.shearwin@adelaide.edu.au
authors.forename: Keith E
authors.initials: KE
authors.lastname: Shearwin
authors.forename: Ian B
authors.initials: IB
authors.lastname: Dodd
authors.forename: J Barry
authors.initials: JB
authors.lastname: Egan
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002874:Chromosome Mapping; D003090:Coliphages / Q000235:genetics; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D019077:Helix-Turn-Helix Motifs; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*; D016335:Zinc Fingers
chemical_list: C107255:CI protein, coliphage 186; D017931:DNA Primers; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins