Figure 1

Organization of the IS 2 insertion sequence and its transposition pathway (modified from [31]). (A) The two-step transposition pathway of IS2. Step I (I) occurs within SC I. Asymmetric single-strand cleavage of the IRR donor is followed by transfer to the donor-inactive IRL recipient end, creating the F-8. Host replication mechanisms convert F-8 into a covalently closed double-stranded circular intermediate, the minicircle. In Step II (II) a second synaptic complex (SC II) is assembled. Cleavage at the abutted CDs results in two exposed 3'OH groups which carry out transesterification attacks on the target DNA. (B) IS2 with IRL (blue) and IRL (red) and two overlapping open reading frames, orfA and orfB, expanded to show detail of the A₆G slippery codons. (i) Translational frameshifting regulates low levels of OrfAB formation; (ii) high levels of the transposase are produced by altering the window to A₇G. (C) Aligned sequences of (i) IRR and (ii) IRL and (iii) the abutted ends of theMCJ. Square brackets identify the termini of IRR and IRL. (i) and (ii): conserved residues (within all elements) are in uppercase; diverged residues (within non-conserved elements A, B, C and I) are in lower case. The extended-10 promoter, P_IRL, (bold underlines identify bases which match the consensus sequence) drives the events of Step I of the transposition pathway shown in panel A. Residues 39 to 48 are shown in these studies to include the binding sequence for the repressor function of Orf A [20]. (iii): abutted ends at the MCJ form a more powerful promoter (P_junc) which indispensably controls the events in Step II. The only functional form of P_junc contains a single base pair spacer (x) which creates its mandatory 17 base pair spacer. CD: cleavage domain; F-8: figure-of-eight; IRR/IRL: right and left inverted repeats; IS: insertion sequence; SC: synaptic complex; MCJ: minicircle junction.