Purification of TnsB, a transposition protein that binds to the ends of Tn7.
We have purified TnsB, a transposition protein encoded by the bacterial transposon Tn7. The purification procedure involves three chromatographic steps (DNA-cellulose, norleucine-Sepharose, and phosphocellulose) and yields milligram quantities of highly purified protein. The apparent molecular mass of denatured TnsB protein is approximately 85 kDa. Gel filtration chromatography and sucrose gradient sedimentation studies indicate that in solution, native TnsB is a monomer of nonspherical shape. Using DNase I protection analysis, we established that TnsB is a sequence-specific DNA-binding protein that recognizes multiple sites in both ends of the transposon. The TnsB binding sites, three in the left end of Tn7 and four in the right end, are highly related in nucleotide sequence and are located in DNA segments that we have previously shown contain cis-acting sequences important for Tn7 transposition. Our results also show that one of the TnsB binding sites overlaps a proposed promoter for the transposition genes of Tn7. These studies suggest that the specific binding of TnsB to the ends of Tn7 mediates recombination and may also regulate the expression of Tn7-encoded transposition genes.
Arciszewska
L K
LK
Department of Microbiology and Immunology, University of California, San Francisco 94143.
McKown
R L
RL
Craig
N L
NL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D012689:Sequence Homology, Nucleic Acid
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C069633:TnsB protein, E coli