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KMID : 0380220070400050740
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Journal of Biochemistry and Molecular Biology
2007 Volume.40 No. 5 p.740 ~ p.748
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Purification and Characterization of Repressor of Temperate S. aureus Phage &pgr;11
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Das Malabika
Ganguly Tridib
Chattoraj Partho
Chanda Palas Kumar
Bandhu Amitava
Lee Chia Yen
Sau Subrata
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Abstract
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To gain insight into the structure and function of repressor proteins of bacteriophages of gram-positive bacteria, repressor of temperate Staphylococcus aureus phage &pgr;11 was undertaken as a model system here and purified as an N-terminal histidine-tagged variant (His-CI) by affinity chromatography. A ~19 kDa protein copurified with intact His-CI (~ 30 kDa) at low level was resulted most possibly due to partial cleavage at its Ala-Gly site. At ~10 nM and higher concentrations, His-CI forms significant amount of dimers in solution. There are two repressor binding sites in &pgr;11 cI-cro intergenic region and binding to two sites occurs possibly by a cooperative manner. Two sites dissected by HincII digestion were designated operators OL and OR, respectively. Equilibrium binding studies indicate that His-CI binds to OR with a little more strongly than OL and binding species is probably dimeric in nature. Interestingly His-CI binding affinity reduces drastically at elevated temperatures (32-42°C). Both OL and OR harbor a nearly identical inverted repeat and studies show that &pgr;11 repressor binds to each repeat efficiently. Additional analyses indicate that &pgr;11 repressor, like &lgr; repressor, harbors an Nterminal domain and a C-terminal domain which are separated by a hinge region. Secondary structure of &pgr;11 CI even nearly resembles to that of &lgr;, phage repressor though they differ at sequence level. The putative N-terminal HTH (helix-turn-helix) motif of &pgr;11 repressor belongs to the HTH -XRE-family of proteins and shows significant identity to the HTH motifs of some proteins of evolutionary distant organisms but not to HTH motifs of most S. aureus phage repressors.
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KEYWORD
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Dimer, Operator, Phi 11, Repressor (CI), Secondary structure and HTH motif
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