Abstract
Aminoacyl-tRNA synthetases (AARSs) couple cognate pairs of amino acids and tRNAs for protein synthesis. The coupling errors can be detrimental, guiding AARS evolution towards high selectivity. To address the limits of the initial amino acid selection, half of AARSs acquired the editing domain to clear the non-cognate substrates that evaded the synthetic site rejection. While high selectivity of the synthetic site is well-established, mechanisms that shaped selectivity of the editing domain remain unknown. To tackle this question, we used a class I isoleucyl-tRNA synthetase (IleRS) from Escherichia coli as a model enzyme and a broad range of non-cognate amino acids efficiently discriminated at the IleRS synthetic site. We found that the IleRS editing site cleared all tested non-cognate amino acids with rates of 35-65 s-1. Thus, surprisingly, the editing site exhibits broad substrate acceptance not limited to the amino acids that jeopardize translational fidelity. This questions the established paradigm of the synthetic-editing sites reciprocity for the clearance of the non-cognate substrates. The editing domain’s low selectivity against the non-cognate substrates contrasts its exquisite specificity in the cognate amino acid rejection. We demonstrated that the latter, being the main constraint during the domain evolution, is established by the residues that promote negative catalysis through destabilisation of the transition state comprising exclusively the cognate amino acid. Finally, we unveiled that IleRS may utilize its editing domain in trans. This sets IleRS as a unique class I AARS, which operates by the class II AARS editing mechanism.
Significance Statement The faithful protein synthesis is a vital property of the cell as errors in translation can diminish cellular fitness and lead to severe neurodegeneration. Aminoacyl-tRNA synthetases (AARS) translate the genetic code by loading tRNAs with the cognate amino acids. The errors in amino acid recognition are cleared at the AARS editing domain through hydrolysis of misaminoacyl-tRNAs. Here we show that the editing domain of class I AARSs does not select for non-cognate amino acids that jeopardize the AARS fidelity. Instead, it selects against the cognate aminoacyl-tRNA providing a new paradigm wherein safeguarding against misediting constrains the editing evolution. This design allows for the broad substrate acceptance of the editing domain, a feature that is generally beneficial for error-correction systems.
Competing Interest Statement
The authors have declared no competing interest.