Loss of Editing Activity during the Evolution of Mitochondrial Phenylalanyl-tRNA Synthetase

Abstract

Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNA Phe . Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNA Phe , an activity enhanced in active site variants with improved tyrosine recognition. Possible trans editing was investigated in isolated mitochondrial extracts, but no such activity was detected. These data indicate that the mitochondrial protein synthesis machinery lacks the tyrosine proofreading activity characteristic of cytosolic translation. This difference between the mitochondria and the cytosol suggests that either organellar protein synthesis quality control is focused on another step or that translation in this compartment is inherently less accurate than in the cytosol.The aminoacyl-tRNA synthetases (aaRS) 2 are a ubiquitous and essential protein family required for protein synthesis (1-3). Structurally and functionally the aaRSs are divided into two unrelated but biochemically analogous groups, class I and class II (4 -6). The aaRSs attach amino acids to the 3Ј-ends of tRNA containing the corresponding anticodon sequence, and the resulting aminoacyl-tRNAs (aa-tRNAs) are used as substrates for ribosomal translation of mRNA. The accuracy of aa-tRNA synthesis is generally assured by the existence of aaRSs specific for each particular amino acid-tRNA pair. Cognate tRNA recognition and discrimination of non-cognate RNAs are achieved by sequencespecific direct and indirect readout of the numerous combinations of bases present in tRNAs (7-10). The relative structural simplicity and inherent similarity between the amino acid substrates makes their accurate recognition and discrimination more challenging. Although some amino acids such as cysteine and tyrosine are different enough to allow their specific recognition by a particular aaRS (11, 12), others such as valine and isoleucine are less easily distinguished. For example the class I aaRS isoleucyl-tRNA synthetase (IleRS) is only able to poorly discriminate against valine, which has a misactivation rate of about 1:200 compared with the cognate substrate isoleucine. Despite this significant rate of misactivation and misaminoacylation, the accuracy of translation is not compromised because of the existence of an intrinsic proofreading and editing mechanism in IleRS that specifically hydrolyzes both misactivated Val-AMP and misaminoacylated Val-tRNA Ile (13,14). In addition to IleRS, many other class I and class II aaRSs also employ editing to prevent release of non-cognate aa-tRNA and subsequent l...

The Homotetrameric Phosphoseryl-tRNA Synthetase from Methanosarcina mazei Exhibits Half-of-the-sites Activity

“…The discrimination observed is above the overall error rate of protein synthesis of 1 in 10 3 to 10 4 , suggesting strongly that SepRS does not require an editing active site (48). This conclusion is underlined by the observation that, in several class II tRNA synthetases that do perform hydrolytic editing, the rate of noncognate amino acid activation is only 40 -300-fold below that for cognate amino acids (49,50). Finally, the sequence of SepRS does not align well with that of the ␤ subunit of PheRS, which contains the B3/B4 domain known to be responsible for editing.…”

Functional Divergence of a Unique C-terminal Domain of Leucyl-tRNA Synthetase to Accommodate Its Splicing and Aminoacylation Roles

“…Examples of editing aminoacyl-tRNA synthetases from origins that lack editing activities include the human cytoplasmic ProRS, human mitochondrial LeuRS, and yeast mitochondrial PheRS. Each fails to hydrolyze mischarged tRNAs or misactivated adenylates (33)(34)(35). For the human enzymes, it has been proposed that enhanced discrimination within the aminoacylation active site combined with low intracellular concentrations of noncognate amino acids that are below the K m of the enzyme apparently suffice to achieve fidelity levels that maintain mitochondrial and/or cellular functions.…”

Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment

“…An intriguing question is why the editing site of MST1 is dispensable in yeast mitochondria. Mitochondrial phenylalanyltRNA synthetases (PheRSs) also lack a cis-editing domain (30), but their aminoacylation active sites are more selective toward amino acids than that of the cytosolic PheRSs. The increased selectivity for amino acid substrates of the mitochondrial PheRSs reduces the overall rate of misacylation to a tolerable level (31).…”