IS 1-encoded proteins, InsA and the InsA-B′-InsB transframe protein (transposase): Functions deduced from their DNA-binding ability

Abstract

Insertion sequence IS 1 encodes a transframe protein, InsA-B′-InsB, which is produced from two out-of-phase reading frames, insA and B′- insB, by translational frameshifting at a run of adenines. Unless the frameshifting event occurs, the InsA protein is produced from IS 1. We found that cells harboring a plasmid carrying an IS 1 mutant with a single adenine insertion in the run of adenines contained miniplasmids. Cloning and DNA sequencing analyses of the miniplasmids revealed that they had a deletion extending from an inverted repeat (IR) at the left end of IS 1. This indicates that they were generated by IS 1-mediated deletion due to efficient production of the InsA-B′-InsB transframe protein that is IS 1 transposase. Both the InsA protein and transposase were partially purified as a fusion protein with collagen-LacZ by LacZ-specific affinity column chromatography. The InsA ∗ and the collagenolyzed InsA ∗ were found to bind specifically to a 24-bp region within each of the IRs at the ends of IS 1. The transposase Tnp ∗ and the collagenolyzed Tnp ∗ were found to bind to the sequence with or without IR, but preferentially to that with IR. The nonspecific DNA-binding ability of transposase may be involved in recognition of the target DNA, an important process of transposition of IS 1. Both InsA and transposase have the IR-specific DNA binding ability and a common polypeptide segment containing the α-helix-turn-α-helix motif, supporting the previous indication that InsA competes with transposase to bind to IRs and thus becomes a transposition inhibitor. Based on the observations described in this article, we speculate that transposase of IS 1 consists of at least two domains, the N-terminal half, which almost entirely overlaps InsA, and the C-terminal half, which almost entirely overlaps B′-InsB. The frameshifting event adds the latter domain to the former to give the transposase activity recognizing IRs and the target sequence to initiate the transposition reaction.