Substrate and Enzyme Functional Groups Contribute to Translational Quality Control by Bacterial Prolyl-tRNA Synthetase

Abstract

Aminoacyl-tRNA synthetases activate specific amino acid substrates and attach them via an ester linkage to cognate tRNA molecules. In addition to cognate proline, prolyl-tRNA synthetase (ProRS) can activate cysteine and alanine and misacylate tRNAPro. Editing of the misacylated aminoacyl-tRNA is required for error-free protein synthesis. An editing domain (INS) appended to bacterial ProRS selectively hydrolyzes Ala-tRNAPro, whereas Cys-tRNAPro is cleared by a freestanding editing domain, YbaK, through a unique mechanism involving substrate sulfhydryl chemistry. The detailed mechanism of catalysis by INS is currently unknown. To understand the alanine specificity and mechanism of catalysis by INS, we have explored several possible mechanisms of Ala-tRNAPro deacylation via hybrid QM/MM calculations. Experimental studies were also performed to test the role of several residues in the INS active site, as well as various substrate functional groups in catalysis. Our results support a critical role for the tRNA 2′-OH group in substrate binding and catalytic water activation. A role is also proposed for the protein’s conserved GXXXP loop in transition state stabilization and for the main chain atoms of Gly261 in a proton relay that contributes substantially to catalysis.

A Multiple Aminoacyl-tRNA Synthetase Complex That Enhances tRNA-Aminoacylation in African Trypanosomes

“…MCP3, which is part of the MARS complex, has sequence similarity to the Ybak protein (47,49) and to Ybak domains of prokaryote ProRS (66). Ybak functions to prevent misaminoacylation by deacylating mischarged tRNAs (67).…”

p53-Dependent DNA damage response sensitive to editing-defective tRNA synthetase in zebrafish

“…Mistakes of aminoacylation occur with a frequency of less than 1% (3,4), and if not corrected, will result in the insertion of a mischarged amino acid into a growing polypeptide chain at the wrong codon (5)(6)(7). This sort of error is usually corrected by a universal editing mechanism in tRNA synthetases, which hydrolytically removes the mischarged amino acid from the tRNA before the wrong amino acid is inserted into a nascent protein (8)(9)(10)(11)(12)(13)(14).…”

Exclusive Use of trans-Editing Domains Prevents Proline Mistranslation

“…The histidine-tagged proteins were purified using HIS-select nickel resin (Sigma-Aldrich). Wild-type (WT) E. coli alanyl-tRNA synthetase (AlaRS) (16), WT ProRS (17), E. coli K279A ProRS (17), E. coli EF-Tu (18), and E. coli tRNA nucleotidyltransferase (19) were prepared as described previously. Concentrations of E. coli AlaRS, E. coli K279A ProRS, C. crescentus YbaK, C. crescentus ProXp-ala, E. coli EF-Tu, and E. coli tRNA nucleotidyltransferase were determined by the Bradford assay (20).…”