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. Author manuscript; available in PMC: 2022 Apr 29.
Published in final edited form as: Cell. 2021 Mar 25;184(9):2441–2453.e18. doi: 10.1016/j.cell.2021.03.006

Figure 5|. Homing of AvCAST is mediated through conserved attTn7 recognition by TnsD.

Figure 5|

(A) RNA-guided insertion frequency of AvCAST into the pTarget with AvPSP1 and glmS gene at each ΔTniQ/ΔTnsD condition. Data are represented as mean ± SD. (B) Tn7-like machinery-mediated insertion frequency into the pTarget with AvPSP1 and glmS gene at each ΔTniQ or ΔTnsD condition. Data are represented as mean ± SD. (C) AvCAST-mediated single prominent insertion at glmS Tn7 attachment site on plasmid identified by deep sequencing. Purple bar indicates the position by Tn7-like machinery (tnsA, tnsB, tnsC, and tnsD). Blue bar indicates the position by all AvCAST protein components (Tn7-like machinery + TniQ + Cascade). Light blue bar indicates the insertion position on the plasmid harboring an additional 65-bp downstream sequence of A. variabilis glmS. (D) Tn7-like machinery-mediated insertion frequency of AvCAST into a plasmid bearing the A.variabilis glmS gene, a plasmid bearing mutations in the conserved attTn7 site, and a plasmid bearing the E.coli glmS gene. The 30 bp of the C-terminus of glmS, a part of attTn7 identified by a previous study (Mitra et al.), is shown. Base numbering format follows that of the study (End of glmS = +23), and reported essential positions for TnsD recognition are shown in red. Data are represented as mean ± SD. (E) Schematic of in vitro transposition reactions with purified Tn7-like machinery components of AvCAST. (F) Tn7-like machinery and donor requirements for in vitro transposition on E. coli glmS Tn7 attachment site. pInsert was detected by PCR for LE and RE junctions. (G) Tns protein requirements for in vitro transposition to the E.coli glmS Tn7 attachment site. All reactions contained pDonor and pTarget.