Identification of Quaternary Structure and Functional Domains of the CI Repressor from Bacteriophage TP901-1
| Autor: | Margit Pedersen, Leila Lo Leggio, J. Günter Grossmann, Sine Larsen, Karin Hammer |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Gene Expression Regulation
Viral Models Molecular Mitomycin Molecular Sequence Data Repressor Electrophoretic Mobility Shift Assay Genome Viral Bacteriophage Protein structure Structural Biology Lysogenic cycle Bacteriophages Viral Regulatory and Accessory Proteins Amino Acid Sequence Promoter Regions Genetic Protein Structure Quaternary Molecular Biology biology DNA-binding domain biology.organism_classification Protein Structure Tertiary Molecular Weight Repressor Proteins Solutions Temperateness Cross-Linking Reagents Lytic cycle Biochemistry Interaction with host DNA Viral Mutation Chromatography Gel Mutant Proteins Sequence Alignment Protein Binding |
| Zdroj: | University of Copenhagen |
| ISSN: | 0022-2836 |
| DOI: | 10.1016/j.jmb.2007.12.022 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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