Post-transfer editing in vitro and in vivo by the beta subunit of phenylalanyl-tRNA synthetase.
Translation of the genetic code requires attachment of tRNAs to their cognate amino acids. Errors during amino-acid activation and tRNA esterification are corrected by aminoacyl-tRNA synthetase-catalyzed editing reactions, as extensively described for aliphatic amino acids. The contribution of editing to aromatic amino-acid discrimination is less well understood. We show that phenylalanyl-tRNA synthetase misactivates tyrosine and that it subsequently corrects such errors through hydrolysis of tyrosyl-adenylate and Tyr-tRNA(Phe). Structural modeling combined with an in vivo genetic screen identified the editing site in the B3/B4 domain of the beta subunit, 40 angstroms from the active site in the alpha subunit. Replacements of residues within the editing site had no effect on Phe-tRNA(Phe) synthesis, but abolished hydrolysis of Tyr-tRNA(Phe) in vitro. Expression of the corresponding mutants in Escherichia coli significantly slowed growth, and changed the activity of a recoded beta-galactosidase variant by misincorporating tyrosine in place of phenylalanine. This loss in aromatic amino-acid discrimination in vivo revealed that editing by phenylalanyl-tRNA synthetase is essential for faithful translation of the genetic code.
Roy
Hervé
H
Department of Microbiology, The Ohio State University, Columbus, OH 43210-1292, USA.
Ling
Jiqiang
J
Irnov
Michael
M
Ibba
Michael
M
D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D008958:Models, Molecular; D009154:Mutation; D010649:Phenylalanine / Q000378:metabolism; D010652:Phenylalanine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D014176:Protein Biosynthesis; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism
D021122:Protein Subunits; D012360:RNA, Transfer, Phe; D014443:Tyrosine; D010649:Phenylalanine; D014448:Tyrosine-tRNA Ligase; D010652:Phenylalanine-tRNA Ligase