Substrate-mediated Fidelity Mechanism Ensures Accurate Decoding of Proline Codons

Abstract

Aminoacyl-tRNA synthetases attach specific amino acids to cognate tRNAs. Prolyl-tRNA synthetases are known to mischarge tRNA Pro with the smaller amino acid alanine and with cysteine, which is the same size as proline. Quality control in proline codon translation is partly ensured by an editing domain (INS) present in most bacterial prolyl-tRNA synthetases that hydrolyzes smaller Ala-tRNA Pro Aminoacyl-tRNA synthetases (aaRSs) 3 play a pivotal role in the decoding of genetic information by catalyzing the esterification of cognate amino acids onto specific transfer RNAs (tRNAs) in a two-step reaction (1). The first step involves amino acid activation with ATP to form an aminoacyl-adenylate intermediate and concomitant pyrophosphate release. The second step involves aminoacyl transfer to the cognate tRNA via a transesterification reaction. Although aaRSs display high substrate specificity, they often misactivate structurally similar amino acids. If left unchecked, these mistakes lead to errors in protein synthesis, and accumulation of such errors can be deleterious to cells (2, 3).To ensure high fidelity in translation, aaRSs have evolved several proofreading mechanisms (4 -7). In the first type of proofreading, misactivated aminoacyl-adenylates are enzymatically hydrolyzed or selectively released from the active site followed by solvent hydrolysis, in a process termed "pretransfer" editing. Another mechanism of proofreading known as "posttransfer" editing is generally believed to function via the socalled "double-sieve" mechanism (8). The model predicted that, whereas one active site could not completely discriminate Ile and Val, two separate active sites with distinct strategies for recognition could significantly enhance fidelity. The aminoacylation active site of the aaRS would act as a "coarse" sieve for adenylate synthesis, activating the cognate amino acid but also allowing, to a lesser extent, activation of isosteric or smaller amino acids that could fit into the amino acid binding pocket. The second "fine" sieve would selectively bind misactivated amino acids for editing while excluding the original cognate amino acid. Thus, substrates synthesized in the first sieve would be further screened by the second sieve to enhance fidelity. Subsequently, biochemical and structural data validated this steric exclusion mechanism for valyl-tRNA synthetase and isoleucyl-tRNA synthetase (9, 10). Indeed, members of both classes of synthetases are now known to possess a second active site spatially separated from the ancient catalytic core to carry out post-transfer editing. Structures of class I isoleucyl-tRNA synthetase (10), leucyl-tRNA synthetase (11), and valyl-tRNA synthetase (12) reveal the highly conserved connective peptide 1 domain that functions in editing. Class II alanyl-tRNA synthetase (AlaRS) (13, 14), threonyl-tRNA synthetase (15), prolyltRNA synthetase (ProRS) (16,17), and phenylalanyl-tRNA synthetase (18, 19) also possess distinct domains for post-transfer editing. A recent study of AlaRS revealed an ...

Substrate Specificity of Bacterial Prolyl-tRNA Synthetase Editing Domain Is Controlled by a Tunable Hydrophobic Pocket

“…Deacylation of Ala-, Ser-, Cys-, Pro-, and 2-Abu-tRNA Pro was carried out at 25°C, whereas deacylation of Cys-tRNA Pro was carried out at 25°C or 37°C according to published protocols (13,16). Reactions were performed using ϳ1 M aminoacyl-tRNA in 150 mM KPO 4 Pro , the reactions were monitored by precipitating the tRNA on Whatman filter pads followed by scintillation counting (12).…”

“…Preparation of radiolabeled Ala-, Ser-, Cys-, Pro-, and 2-aminobutyric acid (2-Abu)-tRNA Pro was carried out as described (12,16 (28). Following aminoacylation reactions, the substrate aminoacyl-tRNAs were phenol-chloroform-extracted, ethanol-precipitated, and stored at Ϫ80°C for use in deacylation assays.…”

“…5D). Interestingly, efficient deacylation of Cys-tRNA Pro by the I263A variant (k obs ϭ 0.068 Ϯ 0.005 min Ϫ1 ) was observed, and weak deacylation by the L266A variant was also detected (k obs ϭ 0.01 Ϯ 0.001 min Ϫ1 ) at 37°C, the temperature typically used for Cys-tRNA deacylation by YbaK (16). To compare the observed rates for Cys-deacylation with the rates obtained for other substrates at room temperature, cysteine deacylation assays using the I263A and L266A variants were also carried at room temperature (supplemental Fig.…”

“…We recently showed that YbaK uses a unique substrate-assisted mechanism of catalysis involving thiol-specific chemistry to cleave Cys-tRNA Pro (16). We hypothesize that due to the similar sizes of cysteine and proline, the ProRS INS domain could not evolve a substrate binding pocket that hydrolyzed both Alaand Cys-tRNA Pro while excluding Pro-tRNA Pro .…”

“…Some species, including those that lack an INS domain, encode a freestanding domain known as PrdX that hydrolyzes Ala-tRNA Pro (14,15). At least three additional freestanding editing domain homologs of unknown function (YeaK, ProX, and PA2301) have been identified based on sequence similarity to INS and YbaK (16).…”