Synthetic and Editing Mechanisms of Aminoacyl-tRNA Synthetases

Abstract

Aminoacyl-tRNA synthetases (aaRS) ensure the faithful transmission of genetic information in all living cells. The 24 known aaRS families are divided into 2 structurally distinct classes (class I and class II), each featuring a catalytic domain with a common fold that binds ATP, amino acid, and the 3'-terminus of tRNA. In a common two-step reaction, each aaRS first uses the energy stored in ATP to synthesize an activated aminoacyl adenylate intermediate. In the second step, either the 2'- or 3'-hydroxyl oxygen atom of the 3'-A76 tRNA nucleotide functions as a nucleophile in synthesis of aminoacyl-tRNA. Ten of the 24 aaRS families are unable to distinguish cognate from noncognate amino acids in the synthetic reactions alone. These enzymes possess additional editing activities for hydrolysis of misactivated amino acids and misacylated tRNAs, with clearance of the latter species accomplished in spatially separate post-transfer editing domains. A distinct class of trans-acting proteins that are homologous to class II editing domains also perform hydrolytic editing of some misacylated tRNAs. Here we review essential themes in catalysis with a view toward integrating the kinetic, stereochemical, and structural mechanisms of the enzymes. Although the aaRS have now been the subject of investigation for many decades, it will be seen that a significant number of questions regarding fundamental catalytic functioning still remain unresolved.

Aminoacyl-tRNA synthetase evolution and sectoring of the genetic code

“…GlyRS) accurately add amino acids to the 3’-CCA ends of tRNAs. Two distinct protein folds are identified for aaRS, described as class I and class II [1,2]. Class I aaRS have an active site that adenylates an amino acid at a “Rossmannoid” fold of parallel β-sheets before transferring the amino acid to tRNA.…”

“…Although the initial evolution of the genetic code may have involved ribozyme-catalyzed tRNA aminoacylation [20–23], at later stages, tRNAs coevolved with aaRS enzymes that attach amino acids at the tRNA 3’-CCA ends [1,2]. Some aaRS enzymes have the capability to proofread tRNA-aa attachments by moving an improperly joined amino acid from the aaRS synthetic site, where the amino acid is linked to the tRNA 3’-CCA end, to a separate aaRS editing or proofreading site, where the non-cognate amino acid is removed [1,2].…”

“…Some aaRS enzymes have the capability to proofread tRNA-aa attachments by moving an improperly joined amino acid from the aaRS synthetic site, where the amino acid is linked to the tRNA 3’-CCA end, to a separate aaRS editing or proofreading site, where the non-cognate amino acid is removed [1,2]. We make observations about aaRS editing that are not noted in reviews nor, as far as we can discover, in the literature.…”

A continuous assay for monitoring the synthetic and proofreading activities of multiple aminoacyl-tRNA synthetases for high-throughput drug discovery

“…Consequently, the active sites of certain aaRSs can sometimes produce improperly charged products (aa-AMP and aa-tRNA). To maintain the fidelity of tRNA aminoacylation, error prone aaRSs have evolved an editing activity, which specifically hydrolyses incorrectly acylated products, either prior to (pre-transfer editing) or following (post-transfer editing) the transfer step [1]. Among the twenty-one existing aaRSs, ten enzymes possess an editing activity.…”

“…This reaction is carried out in an editing site that is distinct from the synthetic site. In the other three enzymes (i.e., Met, Ser, and LysRS), editing and synthesis occur within the same active site, and the editing reaction takes place prior to transfer of the amino acid (for review see [1,2]).…”