Aminoacyl-tRNA synthetases editing sites

Not all aminoacyl-tRNA synthetases have editing sites. The cysteinyl- and tyrosyl-tRNA synthetases bind the correct substrates so much more tightly than their competitors that they do not need to edit.13,14 Similarly, since the accuracy of transcription of DNA by RNA polymerase is better than the overall observed error rate in protein synthesis at about 1 part in 104, RNA polymerases do not need to edit.15 The same should be true for codon-anticodon interactions on the ribosome. However, it is possible that accuracy has been sacrificed to achieve higher rates in this case, which is analogous to a change from Michaelis-Menten to Briggs-Haldane kinetics, and so an editing step is required.16... 

Most aminoacyl-tRNA synthetases contain editing sites in addition to acylation sites. These complementary pairs of sites fimction as a double sieve to ensure very high fidelity. In general, the acylation site rejects amino acids that are larger than the correct one because there is insufficient room for them, whereas the hydrolytic site cleaves activated species that are smaller than the correct one. 

Figure 13.4 The double sieve analogy for the editing mechanism of the isoleucyl-tRNA synthetase. The active site for the formation of the aminoacyl adenylate can exclude amino acids that are larger than isoleucine but not those that are smaller. On the other hand, a hydrolytic site that is just large enough to bind valine can exclude isoleucine while accepting valine and all the smaller amino acids. (In some enzymes, the hydrolytic site offers specific chemical interactions that enable it to bind isosteres of the correct amino acid as well as smaller amino acids.)...

Threonyl-tRNA synthetase can be incubated with tUNA hr that has been covalently linked with serine (Ser-tRNAThr) the tRNA has been "mischarged." The reaction is immediate a rapid hydrolysis of the aminoacyl-tRNA forms serine and free tRNA. In contrast, incubation with correctly charged Thr-tRNAThr results in no reaction. Thus, threonyl-tRNA synthetase contains an additional functional site that hydrolyzes Ser-tRNA hr but not Thr-tHNA hr This editing site provides an opportunity for the synthetase to correct its mistakes and improve its fidelity to less than one mistake in 10. The results of structural and mutagenesis studies revealed that the editing site is more than 20 A from the activation site (Figure 29.9). This site readily accepts and cleaves Ser-tRNAThr but does not cleave Thr-tRNA hr The discrimination of serine from threonine is relatively easy because threonine contains an extra methyl group a site that conforms to the structure of serine will sterically exclude threonine. 

The two-stage reaction allows for selectivity to operate at two levels that of the amino acid and that of the tRNA. The specificity of the first stage uses the fact that the aminoacyl-AMP remains bound to the enzyme. For example, isoleucyl-tRNA synthetase can form an aminoacyl-AMP of isoleucine or the structurally similar valine. If the valyl moiety is then transferred to the tRNA for isoleucine, it is detected by an editing site in the tRNA synthetase, which then hydrolyzes the incorrectly acylated aminoacyl-tRNA. The selectivity resides in the tRNA, not in the amino acid. 

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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