Figure 1.

Organization of the IS2 insertion sequence and its transposition pathway. (A) Wild type IS2 with left and right inverted repeats (IRL, blue; IRR, red) and the two overlapping open reading frames, orfA and orfB, expanded to show the detail of the A₆G slippery codon window which regulates low levels of OrfAB formation (i). High levels of the transposase (TPase) are produced by altering the window to A₇G (ii). (B)Upper. Aligned sequences of IRR and IRL ((i) and (ii)) with the binding domains (yellow) and color coded catalytic domains. Conserved residues are in uppercase and diverged residues are in lower case. The catalytic domain (CD) of IRL contains an additional G/C base pair that is essential for its role in target function [7]. The E-10 promoter, P_IRL, [19] (ii) drives the events of Step I of the transposition pathway [6] resulting in the formation of the minicircle shown in panel C. Lower: Abutted ends at the minicircle junction (MCJ), form a more powerful promoter (P_junc) which indispensably controls the events in Step II of the transposition pathway. The only functional form of P_junc contains a single base pair spacer (x) which creates the mandatory 17 bp spacer. (C) The two-step transposition pathway of IS2. Step I (I) occurs in the TPase-DNA complex, the synaptic complex I (SC I). Asymmetric single strand cleavage of the active IRR donor is followed by strand transfer to the donor-inactive IRL target end, creating the figure-of-eight structure. Host replication mechanisms (HR) convert it into a covalently closed double stranded circular intermediate [10], the minicircle. In step II (II) a second synaptic complex (SC II) is assembled. Cleavages at the abutted CDs result in two exposed 3'OH groups which carry out transesterification attacks on the target DNA. CD: catalytic domain; E-10: extended-10 promoter; IRR/IRL: right and left inverted repeats; IS: insertion sequence; MCJ: minicircle junction; orf: open reading frame; SC: synaptic complex.