Kinetic Origin of Substrate Specificity in Post-Transfer Editing by Leucyl-tRNA Synthetase |
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Authors: | Morana Dulic Nevena Cvetesic Igor Zivkovic Andrés Palencia Stephen Cusack Branimir Bertosa Ita Gruic-Sovulj |
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Institution: | 1. Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia;2. European Molecular Biology Laboratory, Grenoble Outstation and Unit of Virus Host–Cell Interactions, University of Grenoble Alpes–EMBL–CNRS, UMI 3265, Grenoble Cedex 9, France |
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Abstract: | The intrinsic editing capacities of aminoacyl-tRNA synthetases ensure a high-fidelity translation of the amino acids that possess effective non-cognate aminoacylation surrogates. The dominant error-correction pathway comprises deacylation of misaminoacylated tRNA within the aminoacyl-tRNA synthetase editing site. To assess the origin of specificity of Escherichia coli leucyl-tRNA synthetase (LeuRS) against the cognate aminoacylation product in editing, we followed binding and catalysis independently using cognate leucyl- and non-cognate norvalyl-tRNALeu and their non-hydrolyzable analogues. We found that the amino acid part (leucine versus norvaline) of (mis)aminoacyl-tRNAs can contribute approximately 10-fold to ground-state discrimination at the editing site. In sharp contrast, the rate of deacylation of leucyl- and norvalyl-tRNALeu differed by about 104-fold. We further established the critical role for the A76 3′-OH group of the tRNALeu in post-transfer editing, which supports the substrate-assisted deacylation mechanism. Interestingly, the abrogation of the LeuRS specificity determinant threonine 252 did not improve the affinity of the editing site for the cognate leucine as expected, but instead substantially enhanced the rate of leucyl-tRNALeu hydrolysis. In line with that, molecular dynamics simulations revealed that the wild-type enzyme, but not the T252A mutant, enforced leucine to adopt the side-chain conformation that promotes the steric exclusion of a putative catalytic water. Our data demonstrated that the LeuRS editing site exhibits amino acid specificity of kinetic origin, arguing against the anticipated prominent role of steric exclusion in the rejection of leucine. This feature distinguishes editing from the synthetic site, which relies on ground-state discrimination in amino acid selection. |
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Keywords: | aaRS aminoacyl-tRNA synthetase aa-AMP aminoacyl-adenylate aa-tRNA aminoacylated tRNA A76 terminal adenosine of the tRNA aa-tRNA:LeuRS noncovalent complex of aa-tRNA and LeuRS CP1 connective peptide 1 IleRS isoleucyl-tRNA synthetase ITC isothermal titration calorimetry Leu2AA 2′-(L-leucyl)amino-2′-deoxyadenosine Leu2A 2′-L-leucyladenosine Leu-AMS leucyl-adenylate sulphamoyl analogue LeuRS leucyl-tRNA synthetase MD molecular dynamics MST microscale thermophoresis Nva norvaline Nva2AA 2′-(L-norvalyl)amino-2′-deoxyadenosine Nva2A 2′-L-norvalyladenosine ValRS valyl-tRNA synthetase WT wild-type aminoacyl-tRNA synthetases proofreading catalytic RNA substrate-assisted catalysis norvaline |
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