Mechanistic insights into cognate substrate discrimination during proofreading in translation

Abstract

Editing/proofreading by aminoacyl-tRNA synthetases is an important quality control step in the accurate translation of the genetic code that removes noncognate amino acids attached to tRNA. Defects in the process of editing result in disease conditions including neurodegeneration. While proofreading, the cognate amino acids larger by a methyl group are generally thought to be sterically rejected by the editing modules as envisaged by the "DoubleSieve Model." Strikingly using solution based direct binding studies, NMR-heteronuclear single quantum coherence (HSQC) and isothermal titration calorimetry experiments, with an editing domain of threonyl-tRNA synthetase, we show that the cognate substrate can gain access and bind to the editing pocket. High-resolution crystal structural analyses reveal that functional positioning of substrates rather than steric exclusion is the key for the mechanism of discrimination. A strategically positioned "catalytic water" molecule is excluded to avoid hydrolysis of the cognate substrate using a "RNA mediated substrate-assisted catalysis mechanism" at the editing site. The mechanistic proof of the critical role of RNA in proofreading activity is a completely unique solution to the problem of cognate-noncognate selection mechanism.aminoacyl-tRNA synthetases | editing | double-sieve model | enzyme mechanism | X-ray crystallography

E1-E2 Interactions in Ubiquitin and Nedd8 Ligation Pathways

“…These results require that, independent of any contribution to E2 binding, the ␤-grasp domains define carrier protein specificity by excluding non-cognate paralogs. The importance of specificity by binding exclusion rather than affinity is being increasingly appreciated, most recently in accounting for the substrate specificity of caspase-3 versus caspase-9 (49) and editing/proofreading by aminoacyl-tRNA synthetases (50).…”

Transfer RNA: A dancer between charging and mis-charging for protein biosynthesis

“…Recent biochemical and biophysical studies reveal that this DTD-like editing domain of archaeal ThrRS could bind cognate Thr-tRNA Thr , and functional positioning of substrates rather than steric exclusion is the key for the mechanism of discrimination. A properly positioned catalytic water molecule facilitates the hydrolysis of Ser-tRNA Thr while the binding of the cognate Thr-tRNA Thr excludes it, leading to the failure in hydrolysis of correct charged tRNA Thr [74]. This finding improves our understanding of the double-sieve mechanism.…”

“…For example, the editing domain of ThrRS from Pyrococcus abyssi could accommodate cognate Thr in addition to non-cognate Gly and Ser. However, the binding of Thr-tRNA Thr would exclude the catalytically active water which is essential for hydrolysis [74].…”

Universal pathway for posttransfer editing reactions: Insights from the crystal structure of Tt PheRS with puromycin

“…The crystal structure of the editing domain from ThrRS complexed with Ser-A76 reveals two water molecules located on either side of the hydrolyzed bond (9). This study underlines the crucial role played by tRNA in substrateassisted catalysis, in positioning the catalytic water molecules along with the protein side chains (9,10).…”