Aminoacyl-tRNA synthetases proofreading

The tRNA synthetases may provide a check to make sure that the correct amino acid has been attached to the correct tRNA. If an incorrect amino acid is attached to the tRNA, it will be incorporated into the protein at the position specified by the identity of the tRNA. At least some of the aminoacyl tRNA synthetases have a proofreading function that hydrolyzes any incorrect aminoacyl tRNAs (for example, a Val residue attached to an lie tRNA). 

Proofreading by Aminoacyl-tRNA Synthetases The amino-acylation of tRNA accomplishes two ends (1) activation of an amino acid for peptide bond formation and (2) attachment of the amino acid to an adaptor tRNA that ensures appropriate placement of the amino acid in a growing polypeptide. The identity of the amino acid attached to a tRNA is not checked on the ribosome, so attachment of the correct amino acid to the tRNA is essential to the fidelity of protein synthesis. 

In addition to proofreading after formation of the aminoacyl-AMP intermediate, most aminoacyl-tRNA synthetases can also hydrolyze the ester linkage between amino acids and tRNAs in the aminoacyl-tRNAs. This hydrolysis is greatly accelerated for incorrectly charged tRNAs, providing yet a third filter to enhance the fidelity of the overall process. The few aminoacyl-tRNA synthetases that activate amino acids with no close structural relatives (Cys-tRNA synthetase, for example) demonstrate little or no proofreading activity in these cases, the active site for aminoacylation can sufficiently discriminate between the proper substrate and any incorrect amino acid. 

Amino acids are activated by specific aminoacyl-tRNA synthetases in the cytosol. These enzymes catalyze the formation of aminoacyl-tRNAs, with simultaneous cleavage of ATP to AMP and PPj. The fidelity of protein synthesis depends on the accuracy of this reaction, and some of these enzymes carry out proofreading steps at separate active sites. In bacteria, the initiating aminoacyl-tRNA in all proteins is A-formylmethionyl-tRNAfMet. 

J. J. Hopfield has suggested a general mechanism called kinetic proofreading in which there is no hydrolytic site on the enzyme instead, the desired intermediates diffuse into solution, where they hydrolyze nonenzymatically.54 An example is in the selection of amino acids by the aminoacyl-tRNA synthetases (equation 13.32). 

Proofreading by Aminoacyl-tRNA Synthetases Increases the Fidelity of Protein Synthesis... 

Because of the structural similarity between isoleucine and valine, the aminoacyl-tRNA synthetases that link them to their respective tRNAs possess proofreading sites. Examine the structures of the other a-amino acids and determine other sets of amino acids whose structural similarities might also require proofreading. 

The aminoacyl-tRNA synthetases correctly attach each amino acid to its cognate tRNA and proofread the product. 

When errors in ammo acid-tRNA binding do occur, they are usually the result of similarities in amino acid structure. Several aminoacyl-tRNA synthetases possess a separate proofreading site that binds incorrect aminoacyl-tRNA products and hydrolyzes them. 

Because some amino acids are so similar structurally, aminoacyl-tRNA synthetases sometimes make mistakes. These are corrected, however, by the enzymes themselves, which have a proofreading activity that checks the fit In their amino acid-binding pocket. If the wrong amino acid becomes attached to a tRNA, the bound synthetase catalyzes removal of the amino acid from the tRNA. This crucial function helps guarantee that a tRNA delivers the correct amino acid to the protein-synthesizing machinery. The overall error rate for translation In E. coli Is very low, approximately 1 per... 

Aminoacyl tRNA-synthetases have a proofreading ability to double-check that an amino acid is linked with its proper tRNA. The proofreading ability of the enzyme and other proofreading steps (see here) in translation reduce the error frequency to less than 1 in 10,000. 

What is the "second genetic code" The second genetic code refers to the specificity with which the aminoacyl-tRNA synthetases put the correct amino acid onto the correct tRNA. There is only minimal proofreading past that point, so the correct loading of amino acids is critical. If the wrong amino acid is loaded on the tRNA, it is quickly hydrolyzed by the synthetase to avoid such errors. 

Proofreading in amino acid activation takes place in two stages. The first requires a hydrolytic site on the aminoacyl-tRNA synthetase incorrect amino acids that have become esterified to the tRNA are removed here. The second stage of proofreading requires the recognition site on the aminoacyl-tRNA synthetase for the tRNA itself. The incorrect tRNA does not bind tightly to the enzyme. 

Threonyl-tRNA synthetase has a proofreading mechanism. Any Ser-tRNA that is mistakenly formed is hydrolyzed by an editing site 20 A from the activation site. The decision to hydrofyze the aminoacyl-tRNA appears to depend on the size of the amino acid substituent, ff it is smaffer than the correct amino acid, the amino acid fits into the hydrolytic site and is cleaved. If it is the same size as the correct amino acid, it does not fit and is not destroyed. Discrimination between amino acids that are larger than the correct one or are not isoelectronic with it occurs at the aminoacylation step. 

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