Binding of the IS903 transposase to its inverted repeat in vitro.
We have purified the transposase of IS903 in three different ways. We find that transposase expressed as a fusion protein with either glutathione-S-transferase or maltose-binding protein is soluble and can be purified rapidly using affinity chromatography. The third purification requires extracting the native transposase from an insoluble pellet using an alkaline pH buffer. All three proteins bind specifically to the ends of IS903 and give identical patterns of protection when challenged with DNase I. We have used the more stable fusion proteins to examine transposase--DNA interactions in vitro. Methylation interference experiments have identified critical bases for transposase binding; methylated purines that inhibit binding all lie within the inner part of the 18 bp inverted repeat (bp 7-16). Moreover, the positions and identities of these purines suggest that the transposase interacts with base pairs in adjacent major and minor grooves. Binding assays with mutant inverted repeats confirm that transposase binding is sensitive to sequence changes only within this inner region. We propose that the transposase binding site is limited to this domain of the inverted repeat. These data are consistent with our previous analysis of the behaviour of mutant ends in vivo, from which we postulated that the inverted repeat was composed of two functional domains; an inner binding domain (bp 6-18), which included a region of minor groove interactions, and an outer domain that was involved in a step subsequent to transposase binding.
Derbyshire
K M
KM
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06510.
Grindley
N D
ND
D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics; D007446:Chromosome Inversion; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D008745:Methylation; D008780:Methyltransferases; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D019895:Transposases
D002352:Carrier Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D008780:Methyltransferases; D009713:Nucleotidyltransferases; D019895:Transposases