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

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.