Inhibition of transpositional recombination by OrfA and OrfB proteins encoded by insertion sequence IS3.

Abstract

An insertion element IS3 is flanked by terminal inverted repeat (IR) sequences. IS3 encodes two, out-of-phase, overlapping open reading frames, orfA and orfB, from which three proteins are produced. OrfAB is a transframe protein produced by -1 translational frameshifting between orfA and orfB, and it is known to be IS3 transposase. OrfA and OrfB are the proteins produced without frameshifting, but their functions have not been elucidated. A plasmid carrying an IS3 mutant that produces only transposase generates miniplasmids--which are the IS3-mediated intramolecular transposition products--as well as characteristic IS3 circles and linear IS3 molecules. OrfA inhibited the generation of these small molecules to a lesser degree, but OrfB did not. OrfB, together with OrfA, however, inhibited the generation more strongly than OrfA alone. OrfA also inhibited the intermolecular transposition of mini-IS3 with the chloramphenicol-resistance gene flanked by IRs to a reduced frequency, and OrfB together with OrfA inhibited it almost completely. OrfA and/or OrfB did not, however, repress transcription from the promoter in the left-terminal region preceding orfA. The results obtained above show that OrfA and OrfB are not repressors but are inhibitors of transpositional recombination promoted by transposase. OrfA with an alpha helix-turn-alpha helix DNA-binding motif may compete with transposase to bind to terminal IRs. OrfA, together with OrfB that has a DDE motif conserved in retroviral integrases, may inhibit the formation of an active transpososome consisting oftransposase, two terminal IRs and target DNA for the strand transfer reaction. IS3 with a limited size, 1258 bp in length, uses strategies of translational frameshifting and coupling to produce transposase as well as negative regulators to make its copies at a low level, which minimizes a deleterious effect of transposition on bacterial hosts.