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. 2022 Aug 1;119(32):e2202590119. doi: 10.1073/pnas.2202590119

Fig. 1.

Fig. 1.

TnsB STC is a C2-symmetric tetramer with highly intertwined architecture. (A) Schematic of the TnsB transposition mechanism: transposon ends with flanking DNA (green), 5-bp target site (brown), and target DNA (blue). For simplicity, TnsB protomers are not shown. (B) Symmetric strand-transfer DNA substrate is designed to mimic the product of the strand-transfer reaction. The 45-bp part of the transposon left end (LE) includes 1.75 TnsB binding sites (one full binding site, corresponding to 22 bp of L1, and three-fourths of a binding site, corresponding to 16 bp of L2). TnsB binding sites overlap by 1 bp, indicated by green triangles. An 8-bp terminal sequence is colored gray. (C) Domain diagram of TnsB (purple; domain naming conventions follow MuA) and residue positions built for the two different conformations observed: B-L1 and B-L2 (explained further in D). (D) Cryo-EM reconstruction of the TnsB STC. TnsB subunits are referred to according to which TnsB repeat it binds at the transposon end, numbered starting from the target–donor junction. B-L1 is the TnsB monomer that binds the L1 TnsB DNA sequence, and B-L2 binds the L2 site. TnsB monomers are colored shades of purple or orange. B-L1 subunits (light purple and tan) and B-L2 subunits (dark purple and orange) form a C2-symmetric complex. DNA colors are identical with respect to A. (E) Atomic model of the TnsB STC. Two different views are shown; a red asterisk indicates the position of helix IIIα. (F and G) Architectural schematic of (F) the ShTnsB STC and (G) (PDB ID code 4FCY) the MuA STC from bacteriophage Mu. Helix IIIα is indicated with a red asterisk, yellow triangle, or purple square. The location of the DDE catalytic triad is indicated with red stars. Blue stars (DNA-binding domains) indicate domains that are present in MuA but not observed in the ShCAST TnsB STC structure. (H and I) Close-up view of intersubunit interactions in the TnsB STC. (H) Domain IIIα (indicated with a red asterisk) from B-L2 (residues 505 to 519) mediates a three-way interaction in between two B-L1 subunits. (I) B-L1 and B-L2 subunits form stabilizing beta-sheet interactions. Hydrogen bonds are indicated with dashed lines.