Conformational communication mediates the reset step in t6A biosynthesis

Abstract

The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC–AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB2D2E2) bound to Mg2+-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t6A cycle. Further, SAXS analysis of the TmTsaB2D2-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC–AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system.

The structure of the TsaB/TsaD/TsaE complex reveals an unexpected mechanism for the bacterial t6A tRNA-modification

“…The AMPCPP occupies exactly the same position as the carboxy-AMP compound present in the recent structure of Tm TsaBDE, reported by Swairjo et al. (1). We further confirm that in our structure the N-terminal part of the Tm TsaD active site remains partially disordered and that neither Zn nor Fe ions are bound.…”

“…The new structure does not alter the primary conclusions of our manuscript:The C-terminal part of the active site of Tm TsaD remains well-structured and is still capable of binding a nucleotide in the context of the ternary Tm TsaBDE complex.The AMPCPP occupies exactly the same position as the carboxy-AMP compound present in the recent structure of Tm TsaBDE, reported by Swairjo et al. (1). We further confirm that in our structure the N-terminal part of the Tm TsaD active site remains partially disordered and that neither Zn nor Fe ions are bound.…”

Selective inhibition of human translation termination by a drug-like compound

“…2b , 3d –f, 4 , and 5 ) sufficient to visualize chemical modifications of rRNA and tRNA nucleotides (Supplementary Fig. 6 ) 14 – 16 . In the resulting maps of this state, the small-molecule PF846 is also well resolved, along with the protein NC, P-site tRNA, and eRF1 (Fig.…”

Selective inhibition of human translation by a drug-like compound that traps terminated protein nascent chains on the ribosome

“…2b), sufficient to visualize chemical modifications of ribosomal RNA (rRNA) and tRNA nucleotides (Extended Data Fig. 3) [14][15][16] . In the resulting maps, the small molecule PF846 is also well resolved, along with the protein nascent chain, P-site tRNA and eRF1 (Figs.…”

“…The cryo-EM maps have been deposited with the Electron Microscopy Data Bank under the accession codes EMD-____ (non-rotated RNC with eRF1) and EMD-____ Li et al 34 Li et al Li et al 16 Extended Data Table 1 |…”