Identification of Quaternary Structure and Functional Domains of the CI Repressor from Bacteriophage TP901-1

The bacteriophage-encoded repressor protein plays a key role in determining the life cycle of a temperate phage following infection of a sensitive host. The repressor protein Cl, which is encoded by the temperate lactococcal phage TP901-1, represses transcription from both the lytic promoter P-L and the lysogenic promoter PR by binding to multiple operator sites on the DNA. In this study, we used a small bistable genetic switch element from phage TP901-1 to study the effect of cI deletions in vivo and showed that 43 amino acids could be removed from the C-terminal end of Cl without destroying the ability of Cl to repress transcription from the P-L or the bistable switch properties. We showed that a helix-turn-helix motif located in the N-terminal part of CI is involved in DNA binding by introducing specific point mutations. Purification of Cl and truncated forms of Cl followed by analytical gel filtration and chemical cross-linking demonstrated that the C-terminal end of Cl was required for oligomerization and that Cl may exist as a hexamer in solution. Furthermore, expression and purification of the C-terminal part of CI (amino acids 92-180) showed that this part of the protein contained all the amino acids required to form an oligomer with an apparent molecular weight corresponding to a hexamer. We found that the C-terminal end of CI was required for de-repression of the PL following SOS induction, suggesting that the hexameric form of CI is needed for this or that this part of the protein is involved in the interaction with host proteins. By using small-angle X-ray scattering, we show for the first time the overall solution structure of a full-length wild-type bacteriophage repressor at low resolution revealing that the TP901-1 repressor forms a flat oligomer, most probably a trimer of dimers.