Anticodon loop remodeling and D-stem shape drive the specific recognition of ANN-decoding tRNAs for tA modification
Source: PubMed Central Open Access, NCBI / U.S. National Library of Medicine
Abstract N6-threonylcarbamoyladenosine (tA) is a universal transfer RNA (tRNA) modification essential for translational fidelity. The modification is installed at position 37 of ANN-decoding tRNAs (N is A, U, G, or C) by transfer of a threonylcarbamoyl moiety from a pathway intermediate, catalyzed in bacteria by the TsaBD complex. Despite the strict requirement for the 36-UAA-38 sequence in substrate tRNAs, the structural basis for this specificity has remained unclear. We determined cryo-EM structures of thetA synthase bound to unmodified and to natively modified tRNA carrying the tA37 modification, at a nominal resolution of 3.2 Å. In both structures, A37 is positioned in the active site, and the anticodon loop is remodeled into a zig-zag conformation not previously observed in tRNA, stabilized by conserved interactions with the RNA backbone at the TsaD/TsaB interface. The wobble and middle anticodon bases occupy shallow surface pockets without base-specific contacts, explaining tolerance to base identity at these positions. In contrast, U36 and A38 are recognized indirectly through formation of a base triple with U32. Additional D-stem contacts provide a second mode of indirect readout. Together with mutagenesis data, the structures reveal the molecular basis for restriction of tA37 to ANN-decoding tRNAs and the requirement for A38. Graphical Abstract Graphical Abstract For image description, please refer to the figure legend and surrounding text. http://www.w3.org/1999/xl
Abstract
Abstract N6-threonylcarbamoyladenosine (tA) is a universal transfer RNA (tRNA) modification essential for translational fidelity. The modification is installed at position 37 of ANN-decoding tRNAs (N is A, U, G, or C) by transfer of a threonylcarbamoyl moiety from a pathway intermediate, catalyzed in bacteria by the TsaBD complex. Despite the strict requirement for the 36-UAA-38 sequence in substrate tRNAs, the structural basis for this specificity has remained unclear. We determined cryo-EM structures of thetA synthase bound to unmodified and to natively modified tRNA carrying the tA37 modification, at a nominal resolution of 3.2 Å. In both structures, A37 is positioned in the active site, and the anticodon loop is remodeled into a zig-zag conformation not previously observed in tRNA, stabilized by conserved interactions with the RNA backbone at the TsaD/TsaB interface. The wobble and middle anticodon bases occupy shallow surface pockets without base-specific contacts, explaining tolerance to base identity at these positions. In contrast, U36 and A38 are recognized indirectly through formation of a base triple with U32. Additional D-stem contacts provide a second mode of indirect readout. Together with mutagenesis data, the structures reveal the molecular basis for restriction of tA37 to ANN-decoding tRNAs and the requirement for A38. Graphical Abstract Graphical Abstract For image description, please refer to the figure legend and surrounding text. http://www.w3.org/1999/xlink float portrait gkag599figgra1.jpg float ga1 portrait graphical