MOLECULAR-GENETIC ANALYSIS OF TRANSPOSASE-END DNA-SEQUENCE RECOGNITION - COOPERATIVITY OF 3 ADJACENT BASE-PAIRS IN SPECIFIC INTERACTION WITH A MUTANT TN5 TRANSPOSASE

Transposition of Tn5 and IS50 requires the specific binding of transpo sase (Tnp) to the end inverted repeats, the outside end (OE) and the i nside end (IE). OE and IE have 12 identical base-pairs and seven non-i dentical base-pairs. Previously we described the isolation of a Tnp mu tant, EK54, that shows an altered preference for OE versus IE compared to wild-type (wt) Tnp. EK54 enhances OE recognition and decreases IE recognition both in DNA binding and in overall transposition. Here we report that base-pairs 10, 11 and 12 of the OE are critical for the sp ecific recognition by EK54 Tnp. These three adjacent base-pairs act co operatively; all three must be present in order for EK54 Tnp to intera ct very favorably with the end DNA. The existence of only one or two o f these three base-pairs decreases binding of EK54 Tnp. The combined u se of EK54 Tnp and a new OE/IE mosaic end sequence containing the OE b ase-pairs 10, 11 and 12 gives rise to an extraordinarily high transpos ition frequency. Just as the Tnp is a multifunctional protein, the nuc leotides in the 19 bp Tn5 ends also affect other functions besides Tnp binding. Furthermore, the fact that we were able to isolate end seque nce variants that transpose at a higher frequency than the natural end s (OE and IE) with wt Tnp reveals yet another way in which the wt tran sposition frequency is depressed, i.e. by keeping the end sequences su boptimal. (C) 1998 Academic Press Limited.