CIS PREFERENCE OF THE IS903 TRANSPOSASE IS MEDIATED BY A COMBINATION OF TRANSPOSASE INSTABILITY AND INEFFICIENT TRANSLATION

The transposase protein encoded by the insertion element IS903 belongs to an unusual class of DNA-binding proteins, termed cis-acting protei ns, that act preferentially at their site of synthesis, Previous work had led us to propose that instability of the IS903transposase was a m ajor determinant of its cis preference. Here we describe the isolation of two classes of mutations within the transposase gene that increase d action in trans. One class specifically increased trans action witho ut increasing the level of transposition when the mutant gene was loca ted in cis to the transposon. In particular, a threonine-to-proline su bstitution at amino acid 25 (T25P) reduced cis preference about 60-fol d. The half-life of this mutant transposase was significantly longer t han that of the wild-type transposase, confirming the critical role of protein instability. The second, larger, class of mutations increased the level of transposition both in trans and in cis. The behaviour an d location of these mutations were consistent with an increase in gene expression by improving translational initiation. Several of these mu tations exerted a disproportionate effect on the action of transposase in trans, implying that translation efficiency may affect more than j ust the amount of transposase made. Our results indicate that cis pref erence of the IS903 transposase is mediated by a combination of transp osase instability and inefficient translation initiation.