Mechanism of tRNA-dependent editing in translational quality control

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 104; no. 1; pp. 72 - 77
• Main Authors: Ling, Jiqiang, Roy, Hervé, Ibba, Michael
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 02.01.2007; National Acad Sciences
• Subjects: Amino acids; Binding Sites; Biochemistry; Biological Sciences; Catalysis; Enzymes; Esters; Hydrolysis; Hydroxyls; Kinetics; Molecules; Phenylalanine-tRNA Ligase - chemistry; Phenylalanine-tRNA Ligase - physiology; Protein Biosynthesis; Protein synthesis; Ribonucleic acid; RNA; RNA Editing; RNA, Transfer, Phe - chemistry; RNA, Transfer, Phe - physiology; Structure-Activity Relationship; Transfer RNA
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0606272104

Protein synthesis requires the pairing of amino acids with tRNAs catalyzed by the aminoacyl-tRNA synthetases. The synthetases are highly specific, but errors in amino acid selection are occasionally made, opening the door to inaccurate translation of the genetic code. The fidelity of protein synthesis is maintained by the editing activities of synthetases, which remove noncognate amino acids from tRNAs before they are delivered to the ribosome. Although editing has been described in numerous synthetases, the reaction mechanism is unknown. To define the mechanism of editing, phenylalanyl-tRNA synthetase was used to investigate different models for hydrolysis of the noncognate product Tyr-tRNAPhe. Deprotonation of a water molecule by the highly conserved residue βHis-265, as proposed for threonyl-tRNA synthetase, was excluded because replacement of this and neighboring residues had little effect on editing activity. Model building suggested that, instead of directly catalyzing hydrolysis, the role of the editing site is to discriminate and properly position noncognate substrate for nucleophilic attack by water. In agreement with this model, replacement of certain editing site residues abolished substrate specificity but only reduced the catalytic efficiency of hydrolysis 2- to 10-fold. In contrast, substitution of the 3'-OH group of tRNAPhe severely impaired editing and revealed an essential function for this group in hydrolysis. The phenylalanyl-tRNA synthetase editing mechanism is also applicable to threonyl-tRNA synthetase and provides a paradigm for synthetase editing.