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 terminal inverted repeat (IR) of the bacterial transposon gamma delta, a member of the Tn3 family of transposable elements. The effect of the mutant 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 stretch of DNA, running from G8 and A9 to G38; mutations in the outermost seven or eight base-pairs did not significantly affect immunity. Positions at which mutations disrupted immunity chiefly coincided with positions previously determined to constitute three segments of the IR with which gamma delta tranposase protein interacts via major groove contacts. We conclude that sequence-specific binding contacts between gamma delta transposase and its cognate IR are limited to a specific subset of positions (those sensitive to mutation in the immunity assay) within this 30 base-pair region. We found that the innermost of the three major groove contact regions was the most susceptible to mutation, while the outermost was the least. Indications of minor groove contacts were also found. Very few point mutations within the 30 base-pair sequence-specific binding region had much effect on transposition when the mutant ends were in the "wild-type" context with the adjacent integration host factor (IHF) binding site. However, deletion of the IHF site, in some cases, revealed a transposition defect, suggesting that for transposition (but not immunity), IHF-transposase cooperation can largely overcome the effects of reduced transposase binding. Although the 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 transposition that follows transposase binding.
May
E W
EW
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
Grindley
N D
ND
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D019895:Transposases
D004247:DNA; D009713:Nucleotidyltransferases; D019895:Transposases