Functional organization of the inverted repeats of IS30

The mobile element IS30 has 26-bp imperfect terminal inverted repeats (IRs) that are indispensable for transposition. We have analyzed the effects of IR mutations on both major transposition steps, the circle formation and integration of the abutted ends, characteristic for IS30. Several mutants show strikingly different phenotypes if the mutations are present at one or both ends and differentially influence the transposition steps. The two IRs are equivalent in the recombination reactions and contain several functional regions. We have determined that positions 20 to 26 are responsible for binding of the N-terminal domain of the transposase and the formation of a correct 2-bp spacer between the abutted ends. However, integration is efficient without this region, suggesting that a second binding site for the transposase may exist, possibly within the region from 4 to 11 bp. Several mutations at this part of the IRs, which are highly conserved in the IS30 family, considerably affected both major transposition steps. In addition, positions 16 and 17 seem to be responsible for distinguishing the IRs of related insertion sequences by providing specificity for the transposase to recognize its cognate ends. Finally, we show both in vivo and in vitro that position 3 has a determining role in the donor function of the ends, especially in DNA cleavage adjacent to the IRs. Taken together, the present work provides evidence for a more complex organization of the IS30 IRs than was previously suggested.