Purification and characterisation of the TnsB protein of Tn7: a transposition protein that binds to the ends of Tn7.
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.
Tang
Y
Y
Department of Biochemistry, Imperial College of Science, Technology & Medicine, London, UK.
Lichtenstein
C
C
Cotterill
S
S
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C069633:TnsB protein, E coli; D003850:Deoxyribonuclease I