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J. Biol. Chem., Vol. 277, Issue 5, 3186-3194, February 1, 2002

This Article Full Text Full Text (PDF) All Versions of this Article: 277/5/3186    most recent M107740200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shearwin, K. E. Articles by Egan, J. B. Search for Related Content PubMed PubMed Citation Articles by Shearwin, K. E. Articles by Egan, J. B. Social Bookmarking                      What's this?

The Helix-Turn-Helix Motif of the Coliphage 186 Immunity Repressor Binds to Two Distinct Recognition Sequences^*

Keith E. Shearwin, Ian B. Dodd, and J. Barry Egan

From the Department of Molecular Biosciences, University of Adelaide, Adelaide, Australia 5005

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  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.

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