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Publication Abstract from PubMed

The 2-thiouridine (s2U) at the wobble position of certain bacterial and eukaryotic tRNAs enhances aminoacylation kinetics, assists proper codon-anticodon base pairing at the ribosome A-site, and prevents frameshifting during translation. By mass spectrometry of affinity-purified native Escherichia coli tRNA1GlnUUG, we show that the complete modification at the wobble position 34 is 5-carboxyaminomethyl-2-thiouridine (cmnm5s2U). The crystal structure of E. coli glutaminyl-tRNA synthetase (GlnRS) bound to native tRNA1Gln and ATP demonstrates that cmnm5s2U34 improves the order of a previously unobserved 11-amino-acid surface loop in the distal beta-barrel domain of the enzyme and imparts other local rearrangements of nearby amino acids that create a binding pocket for the 2-thio moiety. Together with previously solved structures, these observations explain the degenerate recognition of C34 and modified U34 by GlnRS. Comparative pre-steady-state aminoacylation kinetics of native tRNA1Gln, synthetic tRNA1Gln containing s2U34 as sole modification, and unmodified wild-type and mutant tRNA1Gln and tRNA2Gln transcripts demonstrates that the exocyclic sulfur moiety improves tRNA binding affinity to GlnRS 10-fold compared with the unmodified transcript and that an additional fourfold improvement arises from the presence of the cmnm5 moiety. Measurements of Gln-tRNAGln interactions at the ribosome A-site show that the s2U modification enhances binding affinity to the glutamine codons CAA and CAG and increases the rate of GTP hydrolysis by E. coli EF-Tu by fivefold.

Structural and Mechanistic Basis for Enhanced Translational Efficiency by 2-Thiouridine at the tRNA Anticodon Wobble Position.,Rodriguez-Hernandez A, Spears JL, Gaston KW, Limbach PA, Gamper H, Hou YM, Kaiser R, Agris PF, Perona JJ J Mol Biol. 2013 May 28. pii: S0022-2836(13)00332-X. doi:, 10.1016/j.jmb.2013.05.018. PMID:23727144^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.