Structural and Mechanistic Basis for Enhanced Translational Efficiency by 2-Thiouridine at the tRNA Anticodon Wobble Position

摘要: The 2-thiouridine (s(2)U) at the wobble position of certain bacterial and eukaryotic tRNAs enhances aminoacylation kinetics, assists proper codon-anticodon base pairing ribosome A-site, prevents frameshifting during translation. By mass spectrometry affinity-purified native Escherichia coli tRNA1(Gln)UUG, we show that complete modification 34 is 5-carboxyaminomethyl-2-thiouridine (cmnm(5)s(2)U). crystal structure E. glutaminyl-tRNA synthetase (GlnRS) bound to tRNA1(Gln) ATP demonstrates cmnm(5)s(2)U34 improves order a previously unobserved 11-amino-acid surface loop in distal β-barrel domain enzyme imparts other local rearrangements nearby amino acids create binding pocket for 2-thio moiety. Together with solved structures, these observations explain degenerate recognition C34 modified U34 by GlnRS. Comparative pre-steady-state kinetics tRNA1(Gln), synthetic containing s(2)U34 as sole modification, unmodified wild-type mutant tRNA2(Gln) transcripts exocyclic sulfur moiety tRNA affinity GlnRS 10-fold compared transcript an additional fourfold improvement arises from presence cmnm(5) Measurements Gln-tRNA(Gln) interactions A-site s(2)U glutamine codons CAA CAG increases rate GTP hydrolysis EF-Tu fivefold.