CP1-dependent partitioning of pretransfer and posttransfer editing in leucyl-tRNA synthetase

Abstract

Mistranslation is toxic to bacterial and mammalian cells and can lead to neurodegeneration in the mouse. Mistranslation is caused by the attachment of the wrong amino acid to a specific tRNA. Many aminoacyl-tRNA synthetases have an editing activity that deacylates the mischarged amino acid before capture by the elongation factor and transport to the ribosome. For class I tRNA synthetases, the editing activity is encoded by the CP1 domain, which is distinct from the active site for aminoacylation. What is not clear is whether the enzymes also have an editing activity that is separable from CP1. A point mutation in CP1 of class I leucyl-tRNA synthetase inactivates deacylase activity and produces misacylated tRNA. In contrast, although deletion of the entire CP1 domain also disabled the deacylase activity, the deletion-bearing enzyme produced no mischarged tRNA. Further investigation showed that a second tRNA-dependent activity prevented misacylation and is intrinsic to the active site for aminoacylation.amino acid editing ͉ fidelity ͉ protein synthesis

Leucyl-tRNA synthetase editing domain functions as a molecular rheostat to control codon ambiguity in Mycoplasma pathogens

“…coli LeuRS harbors a tRNA-dependent pretransfer editing activity that is unmasked when its CP1 domain is deleted (21). We also found that we could activate pretransfer editing in E. coli LeuRS by incorporating an unchargeable 2′-deoxyadenosine tRNA Leu (2′dA-tRNA) (21).…”

“…coli LeuRS harbors a tRNA-dependent pretransfer editing activity that is unmasked when its CP1 domain is deleted (21). We also found that we could activate pretransfer editing in E. coli LeuRS by incorporating an unchargeable 2′-deoxyadenosine tRNA Leu (2′dA-tRNA) (21). Thus, we hypothesized that the pathway to amino acid editing can shift between pre-and posttransfer editing mechanisms that are inherent to the enzyme (8,25).…”

“…Remarkably, we determined that the addition of the CP1 domain enhances pretransfer editing activity and fidelity for each of these diverse hybrid models. This contrasts with deletion of CP1 editing domains from E. coli and Saccharomyces cerevisiae mitochondria LeuRSs, which activated a latent tRNA-dependent pretransfer editing activity that suppresses tRNA mischarging (21). We hypothesize, then, that the CP1 domain has evolved to function as a molecular rheostat to enhance fidelity by idiosyncratically potentiating substrate specificity (30) while balancing hydrolytic mechanisms that are associated with either the aminoacylation core or the separate editing domain.…”

“…Previously, we showed that we can remove the CP1 domain from yeast mitochondrial and E. coli LeuRS and maintain complete fidelity (21). In these two LeuRS cases, the CP1 domain acted as a rheostat to dampen pretransfer editing.…”

“…Both tRNA-dependent (20)(21)(22)(23) and tRNA-independent (22, 24) pretransfer editing activities have been reported. Also, clearance of the adenylate by selective ejection from the enzyme's active site into the aqueous environment has been suggested (24).…”