Extended Data Fig. 6: Overlapping region between tnpB gene and ωRNA.
From: Cryo-EM structure of the transposon-associated TnpB enzyme
(a) Schematic illustrating the overlapping region between the tnpB gene (residues 335 to 408) and ωRNA (−231G to −10U). The disordered regions of TnpB and ωRNA are shown as dotted arrows. Except for a few nucleotides (indicated by the red box), the functionally important regions of the tnpB gene and ωRNA do not overlap. (b) In vitro DNA cleavage assay of the wild-type (WT) TnpB, the 5′ region of the ωRNA-deleted (Δ−231G to −117T; Δ5′ region) mutant, and the C-terminal ___domain-deleted (Δ376 to 408; ΔCTD) mutant. The linearized plasmid target, containing a 16-nt target sequence and a TTGAT TAM sequence, was incubated with the TnpB–ωRNA complex at 37 °C for 30 min. The cleavage products were then analyzed by a MultiNA microchip electrophoresis system. (c) Quantification of the DNA cleavage data in (b). Data are mean ± s.d. (n = 3, biologically independent samples). The experiments were repeated three times with similar results. Source data are provided as a Source data file. (d) Thermal shift assay of the WT TnpB and the ΔCTD mutant, calculated by a NanoTemper Tycho NT.6 Differential Scanning Fluorimeter, which determines the inflection temperature (Ti) of samples.
