Functional analysis of the two domains in the terminal inverted repeat sequence required for transposition of Tn3.

Abstract

Bacterial transposon Tn3 has a 38-bp terminal inverted repeat (IR) sequence. The IR sequence has been divided into two domains, A and B, of which domain B is bound by transposase, and domain A is not Here, we defined the two domains more precisely by constructing three IR mutants with a 2-bp substitution at relevant sites within the IR sequence, followed by examination of the binding of transposase to the fragments containing these IR mutants: domain A was located at bp 1-11, whereas domain B was at bp 12-38. To see if the two domains in the IR are functionally distinct, we constructed mini-Tn3 derivatives flanked by two IRs with various 2-bp substitutions within domain A or B, and analyzed their ability to mediate cointegration. The mini-Tn3 derivatives flanked by IR(A+ B+) and IR(A- B+) [or IR(A+ B-)] and those flanked by IR(A-B+) and IR(A+ B-) mediate cointegration more efficiently than the mini-Tn3 derivatives flanked by two IR(A- B+)s or by two IR(A+ B-)s. These results and others presented here indicate that the two domains of IR are functionally distinct in promoting cointegration.