Purification and characterisation of the TnsB protein of Tn7: a transposition protein that binds to the ends of Tn7. |
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Authors: | Y Tang C Lichtenstein and S Cotterill |
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Institution: | Department of Biochemistry, Imperial College of Science, Technology & Medicine, London, UK. |
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Abstract: | Tn7, a large bacterial transposon encodes 5 proteins required for its transposition. We report a rapid and easy purification of one of these proteins, TnsB, from an overexpression strain. This protein was shown to bind to the ends of Tn7, in a bandshift assay, in two distinct stages as a function of protein concentration. DNasel footprinting at each end of Tn7 showed that the TnsB recognition sequence, a set of 22 bp repeats, plus Tn7 termini are protected. Binding of TnsB appeared cooperative but was only observed above a threshold concentration of protein. ATP and Mg2+ had no effect on the pattern of protection, nor did addition of other Tn7-encoded proteins. Hydroxyl radical footprinting, performed at the right end, showed that TnsB binds preferentially to one side of the DNA helix. |
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