Chapter 27 : Copy-out–Paste-in Transposition of IS911: A Major Transposition Pathway

Organization of IS911. (A) Genetic organization. The 1,250-bp IS911 is shown as a box. The boxes at each end represent the left (IRL) and right (IRR) terminal inverted repeats. The two open reading frames, orfA (blue) and orfB (green) are positioned in relative reading phases 0 and −1, respectively, as indicated. The indigenous promoter, pIRL, is shown. The region of overlap between orfA and orfB, which includes the frameshifting signals to produce OrfAB, lies within IS911 coordinates 300 and 400. The precise point at which the frameshift occurs, within the last heptad of the LZ, is indicated by the vertical dotted line. (B) Structure function map of OrfAB and OrfA. HTH, a potential helix-turn-helix motif; LZ, a leucine zipper motif involved in homo- and hetero-multimerization of OrfAB and OrfA. Programmed translational frameshifting that fuses OrfA and OrfB to generate the transposase OrfAB occurs within the fourth heptad. The LZ of OrfA and OrfAB therefore differ in their fourth heptad. A second region, M, necessary for multimerization of OrfAB is shown, as is the catalytic core of the enzyme which carries a third multimerization domain. OrfA translation initiates at an AUG, terminates with UAA whereas OrfAB translation terminates within the right IR. The vertical line to the right of M shows the extent of the truncated transposase, OrfAB[1–149] described in the text. (C) Frameshifting window. The mRNA sequence around the programmed translational frameshifting window is presented. The boxed sequence GGAG is the potential ribosome-binding site located upstream of orfB whose potential translation would be initiated at the boxed AUU codon. A ribosome (not to scale) is shown covering a series of “slippery” codons (AAAAAAG). A downstream secondary structure is also shown with the UAA, OrfA translation termination codon. The ribosome-binding site, slippery codons and secondary structure all contribute to the efficiency of the programmed −1 frameshift. The box at the foot of this figure shows how the anti-codons of two tRNALys are thought to undergo re-pairing with their codons in the AAAAAAG motif.

Important sequence features of IS911. (A) Organization of the IS911 inverted repeat (IR). The nucleotide sequence of IRL and IRR is boxed. Grey horizontal bars above and below indicate the internal regions protected from DNaseI digestion by binding of OrfAB[1–149], a derivative of the 382-amino-acid OrfAB truncated for its catalytic domain. The dotted horizontal grey bar indicates partial protection. The dashes within the sequence indicate mismatches between the left and right ends. The −35 and −10 components of the indigenous promoter pIRL (blue boxes) and of pjunc (green boxes) are shown. The conserved 5′TG tips are highlighted in red. (B) Organization of pjunc. The “junction” promoter assembled on circularization of IS911 is shown as green boxes. The initiating transcript nucleotide (+1 pjunc), the indigenous pIRL (blue boxes) and the initiating transcript nucleotide (+1 pIRL) are also shown. The conserved 5′TG tips are highlighted in red. (C) Secondary structure at the left IS911 end. The sequence of the “top” strand of IRL is shown together with the various transcription and translation signals. The symbols below are standard “dot–bracket” notations to indicate potential secondary structures formed with transcripts from top to bottom: from an external promoter, from pjunc, or from pIRL respectively. The brackets shown in italic simply permit the reader to identify the apical stem of the secondary structure.

Co-translational binding model. This schematic, not to scale, shows the insertion sequence (IS) with its left (IRL) and right (IRR) ends in green. RNA polymerase, RNAP, is shown in pale green in the process of transcribing from the promoter pIRL. The mRNA is shown in dark green with a ribosome (blue) paused at the secondary structure shown in Figure 1C . The nascent OrfAB peptide (brown) is shown binding to IRL while undergoing translation. Above is shown the full-length OrfAB in a folded configuration proposed to prevent its binding to the IR as a completed protein.

The IS911 transpososome. The schematic shows the proposed configuration and composition of the different synaptic complexes (SCA and SCB) involved in different steps of the IS911 transposition cycle. (A) The excision complex SCA. The tips of the insertion sequence (IS), which are not protected by the truncated transposase OrfAB[1–149] ( Figure 2A ) are shown as green circles containing an arrowhead. The inverted repeats (IR) are indicated by thick black lines and the IS as green lines. Full-length OrfAB, which is presumed to cover the entire IR, is shown bound as a monomer to each end and to introduce a small bend in the DNA. Dimerization creates SCA, resulting in pairing of both IRs and in the formation of a DNA loop which includes the IS. Finally, a cleavage and strand transfer event results in the formation of a single-strand bridge between the IRs. (B) The integration complex SCB. Symbols are as in (A). In the left hand column, the IS circle intermediate with its newly replicated strand (dotted line) is shown to form a complex between an IR in the circle and a second in the target to form SCBt. Cleavage and strand transfer is shown to form a single-strand bridge between the two IRs. The RecG helicase is thought to intervene to drive strand migration before a second cleavage and strand transfer results in integration of the circle ( 89 ). This type of mechanism would explain the integration of the many different ISs observed to occur next to a resident IR in the target. The right-hand column shows an untargeted integration event that involves OrfA in addition to OrfAB. It should be emphasized that OrfA is known to interact with OrfAB. It also changes in some way OrfAB binding but it is not clear whether it remains in the complex.

The IS911 transposition cycle. The transposon is shown in green, the flanking donor DNA in black and the target DNA in blue. Transposon ends are shown as green filled circles. The small arrows shown in Figure 4 have been omitted for brevity. (A) Donor plasmid carrying the insertion sequence (IS). (B) Formation of the first synaptic complex SCA and cleavage of the left or right inverted repeat (IR) and attack of the other end. (C) Formation of a single-strand bridge to create a figure-eight molecule if the donor is a plasmid as shown here. (D) The products of IS-specific replication: the double strand circular IS transposition intermediate and the regenerated transposon donor plasmid. The replicated strand is shown as a green dotted line. (E) Formation of the second synaptic complex SCB and engagement of the target DNA (blue). (F) Cleavage of the IS circle and integration. (G) The newly integrated IS.