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

Abstract

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 E. 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 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 β-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 four-fold 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 five-fold.

An evolutionary approach uncovers a diverse response of tRNA 2-thiolation to elevated temperatures in yeast

“…In this assay, thiolated tRNAs are specifically retarded in a polyacrylamide gel (Igloi 1988 -allowing us to quantify all modification targets in parallel. Third, this wobble uridine modification has been implicated in translational control (Grosjean 2009;Rodriguez-Hernandez et al 2013).…”

“…The mcm 5 s 2 U modification is generated through the concerted action of the elongator (ELP) pathway, which is required for formation of the mcm 5 side chain, and the URM1 pathway that is essential for 2-thiolation (s 2 ) (Huang et al 2005(Huang et al , 2008Björk et al 2007;Nakai et al 2008;Schlieker et al 2008;Leidel et al 2009;Noma et al 2009). The physiological role of mcm 5 s 2 U is not well established, although it is thought to stabilize codon-anticodon interactions (Grosjean 2009;Rodriguez-Hernandez et al 2013), and yeast strains lacking U 34 modifications are sensitive to high temperatures and chemical stress (Esberg et al 2006;Leidel et al 2009). …”

Site-selected incorporation of 5-carboxymethylaminomethyl(-2-thio)uridine into RNA sequences by phosphoramidite chemistry

“…TMSE blockage is compatible with the protection of commercially available canonical monomeric units as well as t 6 A, which is a natural modification located at position 37 of yeast mt-ASL Lys . Hypermodified monomers 13a and 13b were effectively incorporated into the anticodon arm sequences of S. cerevisiae mt-tRNA Leu and mt-tRNA Lys , respectively.…”

Celebrating wobble decoding: Half a century and still much is new

“…3). Individual anticodon domain modified nucleosides are identity determinants for protein recognition, particularly aminoacylation, 15-17 increase accuracy and efficiency in codon recognition, 18-24 and pre-structure the ASL for translation. 9,25-30 Each of the modified nucleosides contribute distinct chemistries, nucleoside conformations and dynamics, and their contributions to decoding have been studied extensively over decades and most recently reviewed.…”

“…11 Thus, posttranscriptional modifications of the ASL nucleosides emulate the chemistries of amino acid side chains. 14 Even what appears to be one of the simplest of modifications, such as the substitution of a sulfur atom for an oxygen, a thioketone for a carbonyl group, or the deamination of adenosine to inosine (I), takes on significance in the decoding of mRNA. The modification of wobble position U 34 to s 2 U 34 alters tRNA's ability to wobble to G3; 5 the modification of C 32 to s 2 C 32 negates the ability of tRNAs with I 34 to decode A3 of the codon.…”