The helix-turn-helix motif of the coliphage 186 immunity repressor binds to two distinct recognition sequences.
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.
Shearwin
Keith E
KE
Department of Molecular Biosciences, University of Adelaide, Adelaide, Australia 5005. keith.shearwin@adelaide.edu.au
Dodd
Ian B
IB
Egan
J Barry
JB
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
C107255:CI protein, coliphage 186; D017931:DNA Primers; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins