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 CI, which is encoded by the temperate lactococcal phage TP901-1, represses transcription from both the lytic promoter P(L) and the lysogenic promoter P(R) 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 CI without destroying the ability of CI 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 CI and truncated forms of CI followed by analytical gel filtration and chemical cross-linking demonstrated that the C-terminal end of CI was required for oligomerization and that CI 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 P(L) 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.
Pedersen
Margit
M
BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark. pedersen333@gmail.com
Lo Leggio
Leila
L
Grossmann
J Günter
JG
Larsen
Sine
S
Hammer
Karin
K
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004279:DNA, Viral / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D016679:Genome, Viral; D016685:Mitomycin / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry*; D016415:Sequence Alignment; D012996:Solutions; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry*
D003432:Cross-Linking Reagents; D004279:DNA, Viral; D050505:Mutant Proteins; D012097:Repressor Proteins; D012996:Solutions; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D016685:Mitomycin