IS911 transposition is regulated by protein-protein interactions via a leucine zipper motif

Efficient intermolecular transposition of bacterial insertion sequence IS91 1 involves the activities of two element-encoded proteins: the transposase, OrfAB, land a regulatory factor, OrfA. OrfA shares the majority of its ami no acid sequence with the N-terminal part of OrfAB. This includes a putativ e helix-turn-helix and three of four heptads of a leucine zipper motif. Orf A strongly stimulates OrfAB-mediated intermolecular transposition both in v ivo and in vitro. The present results support the notion that this is accom plished by direct interaction between these two proteins via the leucine zi pper. We used both a genetic approach, based on gene fusions with phage lam bda repressor, and a physical approach, involving coimmunoprecipitation, to show that OrfA not only undergoes oligomerisation but is capable of engagi ng with OrfAB to form heteromultimers, and that the leucine zipper is neces sary for both types of interaction. Furthermore, mutation of the leucine zi pper in OrfA inactivated its regulatory function. Previous observations dem onstrated that the integrity of the leucine zipper motif was also important for OrfAB binding to the IS911 terminal inverted repeats. Here, we show, i n gel shift experiments, using a derivative of OrfAB deleted for the C-term inal catalytic domain, OrfAB[1-149], that the protein is capable of pairing two inverted repeats to generate a species resembling a "synaptic complex" . Preincubation of OrfAB[1-149] with OrfA dramatically reduced formation of this complex and favored formation of an alternative complex devoid of Orf A. Together these results suggest that OrfA exerts its regulatory effect by interacting transiently with OrfAB via the leucine zipper and modifying Or fAB binding to the inverted repeats. (C) 2000 Academic Press.