Abstract
Accurate translation of genetic information into proteins is vital for cell sustainability. ProXp-ala prevents proteome-wide Pro-to-Ala mutations by hydrolyzing misacylated Ala-tRNAPro, which is synthesized by prolyl-tRNA synthetase. Bacterial ProXp-ala was previously shown to combine a size-based exclusion mechanism with conformational and chemical selection for the recognition of the alanyl moiety, whereas tRNAPro is selected via recognition of tRNA acceptor-stem elements G72 and A73. The identity of these critical bases changed during evolution with eukaryotic cytosolic tRNAPro possessing a cytosine at the corresponding positions. The mechanism by which eukaryotic ProXp-ala adapted to these changes remains unknown. In this work, recognition of the aminoacyl moiety and tRNA acceptor stem by human (Homo sapiens, or Hs) ProXp-ala was examined. Enzymatic assays revealed that Hs ProXp-ala requires C72 and C73 in the context of Hs cytosolic tRNAPro for efficient deacylation of mischarged Ala-tRNAPro The strong dependence on these bases prevents cross-species deacylation of bacterial Ala-tRNAPro or of Hs mitochondrial Ala-tRNAPro by the human enzyme. Similar to the bacterial enzyme, Hs ProXp-ala showed strong tRNA acceptor-stem recognition but differed in its amino acid specificity profile relative to bacterial ProXp-ala. Changes at conserved residues in both the Hs and bacterial ProXp-ala substrate-binding pockets modulated this specificity. These results illustrate how the mechanism of substrate selection diverged during the evolution of the ProXp-ala family, providing the first example of a trans-editing domain whose specificity evolved to adapt to changes in its tRNA substrate.
Highlights
Correct pairing of amino acids with their corresponding transfer RNA (tRNA) by aminoacyl-tRNA synthetases (ARSs) is fundamental for faithful translation of genetic information into proteins
Biochemical studies revealed that Cc ProXp-Ala and E. coli (Ec) prolyl-tRNA synthetase (ProRS) share similar size-exclusion and chemical-based mechanisms for recognition of the aminoacyl moiety, limiting their ability to hydrolyze tRNAPro charged with cognate Pro, as well as other amino acids that are larger than Ala
In contrast to a previous report, which concluded that H. sapiens (Hs) cyto ProXp-ala lacks tRNA specificity [31], we found a strong dependence on elements at the top of the acceptor stem for efficient deacylation by the Hs enzyme (Table 1)
Summary
Correct pairing of amino acids with their corresponding tRNAs by aminoacyl-tRNA synthetases (ARSs) is fundamental for faithful translation of genetic information into proteins. Biochemical studies revealed that Cc ProXp-Ala and E. coli (Ec) ProRS share similar size-exclusion and chemical-based mechanisms for recognition of the aminoacyl moiety, limiting their ability to hydrolyze tRNAPro charged with cognate Pro, as well as other amino acids that are larger than Ala
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