A FUNCTIONAL-ANALYSIS OF THE INVERTED REPEAT OF THE GAMMA-DELTA TRANSPOSABLE ELEMENT

We have constructed a library of point mutants of the 35 base-pair ter minal inverted repeat (IR) of the bacterial transposon gamma delta, a member of the Tn3 family of transposable elements. The effect of the m utant ends, both on the immunity conferred on an IR-containing target plasmid and on the transposition of model transposons, was determined. The region important for immunity was shown to be a 30 base-pair stre tch of DNA, running from G8 and A9 to G38; mutations in the outermost seven or eight base-pairs did not significantly affect immunity. Posit ions at which mutations disrupted immunity chiefly coincided with posi tions previously determined to constitute three segments of the IR wit h which gamma delta tranposase protein interacts via major groove cont acts. We conclude that sequence-specific binding contacts between gamm a delta transposase and its cognate IR are limited to a specific subse t of positions (those sensitive to mutation in the immunity assay) wit hin this 30 base-pair region. We found that the innermost of the three major groove contact regions was the most susceptible to mutation, wh ile the outermost was the least. Indications of minor groove contacts were also found. Very few point mutations within the 30 base-pair sequ ence-specific binding region had much effect on transposition when the mutant ends were in the ''wild-type'' context with the adjacent integ ration host factor (IHF) binding site. However, deletion of the IHF si te, in some cases, revealed a transposition defect, suggesting that fo r transposition (but not immunity), IHF-transposase cooperation can la rgely overcome the effects of reduced transposase binding. Although th e outer seven base-pairs were not important for immunity, mutations in the outer three or four eliminated or reduced transposition activity, suggesting that these positions are involved in a step in transpositi on that follows transposase binding.