Extended Data Fig. 1: IS621 insertion sequence element.

(a) Transposition cycle of the IS621 insertion sequence element. The IS621 elements consist of the left end (LE), the recombinase-coding sequence, and the right end (RE), flanked by the CT core dinucleotide sequences at both ends. The transposition cycle of the IS621 elements consists of excision (generation of a circular form) and insertion (recombination between the circular form and genomic target sites) steps. In the excision step, recombination would occur between the 5′ left target (LT)–core–right donor (RD) region and the 3′ left donor (LD)–core–right target (RT) region in the IS621 locus, resulting in the LT–core–RT region in the original genomic site and the LD–core–RD region at the RE–LE junction in the circular form. Importantly, the RE–LE junction contains the reconstituted σ⁷⁰-like promoter sequence, since RE and LE contain a −35 box and a −10 box, respectively². Thus, the bRNA encoded in the LE is expressed downstream of the core sequence at the RE–LE junction in the circular intermediate². However, it remains unclear whether the excision is mediated by the IS621–bRNA complex and, if so, how the IS621–bRNA complex accomplishes both excision and insertion reactions. For our cryo-EM analysis, we used the 44-bp donor DNA (the RE–LE junction with the LD–core–RD sequence in the circular form) and the 38-bp target DNA (the genomic target site with the LT–core–RT sequence) from the natural IS621 element found in E. coli, with the indicated minor modifications to assist structural analysis. 5′ SL, 5′ stem loop; TBL, target-binding loop; DBL, donor-binding loop; LTG, left target guide; RTG, right target guide; LDG, left donor guide; RDG, right donor guide; TS, top strand; BS, bottom strand. (b) Schematics showing base-pairing between the bRNA and tDNA/dDNA. Mismatched (MM) nucleotides introduced to the top strands for the structural analysis are shown as lower-case letters.