The discriminator base N73 is a key identity element of tRNA. In eukaryotes, N73 is an "A" in cytoplasmic tRNA and a "C" in mitochondrial tRNA. We present evidence herein that yeast histidyl-tRNA synthetase (HisRS) recognizes both A73 and C73, but somewhat prefers A73 even within the context of mitochondrial tRNA. In contrast, humans possess two distinct yet closely related HisRS homologues, with one encoding the cytoplasmic form (with an extra N-terminal WHEP domain) and the other encoding its mitochondrial counterpart (with an extra N-terminal mitochondrial targeting signal). Despite these two isoforms sharing high sequence similarities (81% identity), they strongly preferred different discriminator bases (A73 or C73). Moreover, only the mitochondrial form recognized the anticodon as a strong identity element. Most intriguingly, swapping the discriminator base between the cytoplasmic and mitochondrial tRNA isoacceptors conveniently switched their enzyme preferences. Similarly, swapping seven residues in the active site between the two isoforms readily switched their N73 preferences. This study suggests that the human HisRS genes, while descending from a common ancestor with dual function for both types of tRNA, have acquired highly specialized tRNA recognition properties through evolution.
Previous studies showed that valyl-tRNA synthetase of Saccharomyces cerevisiae contains an N-terminal polypeptide extension of 97 residues, which is absent from its bacterial relatives, but is conserved in its mammalian homologues. We showed herein that this appended domain and its human counterpart are both nonspecific tRNA-binding domains (K d ϳ 0.5 M). Deletion of the appended domain from the yeast enzyme severely impaired its tRNA binding, aminoacylation, and complementation activities. This N-domain-deleted yeast valyltRNA synthetase mutant could be rescued by fusion of the equivalent domain from its human homologue. Moreover, fusion of the N-domain of the yeast enzyme or its human counterpart to Escherichia coli glutaminyl-tRNA synthetase enabled the otherwise "inactive" prokaryotic enzyme to function as a yeast enzyme in vivo. Different from the native yeast enzyme, which showed different affinities toward mixed tRNA populations, the fusion enzyme exhibited similar binding affinities for all yeast tRNAs. These results not only underscore the significance of nonspecific tRNA binding in aminoacylation, but also provide insights into the mechanism of the formation of aminoacyl-tRNAs.Aminoacyl-tRNA synthetases are a group of ancient enzymes, each of which catalyzes the attachment of a specific amino acid to its cognate tRNAs. Aminoacyl-tRNAs are then delivered by elongation factor-1 (EF-1) 3 to ribosomes for protein translation. In prokaryotes, there are typically 20 aminoacyl-tRNA synthetases, one for each amino acid (1-4). In eukaryotes, protein synthesis occurs not only in the cytoplasm, but also in organelles, such as mitochondria and chloroplasts (5). Thus, eukaryotes, such as yeast, commonly have two genes that encode distinct sets of proteins for each aminoacylation activity, one localized to the cytoplasm and the other to the mitochondria. Each set aminoacylates the isoaccepting tRNAs within its respective cell compartment and is sequestered from the isoacceptors confined in other compartments. In most cases, cytoplasmic and mitochondrial synthetase activities are encoded by two distinct nuclear genes, regardless of the cell compartments to which they are confined. However, in some cases, cytoplasmic and mitochondrial forms of a tRNA synthetase with a given amino acid specificity are encoded by the same nuclear gene through alternative initiation of translation, examples of which include ALA1 (coding for alanyl-tRNA synthetase) (6, 7), GRS1 (coding for glycyl-tRNA synthetase) (8), HTS1 (coding for histidyl-tRNA synthetase) (9), and VAS1 (coding for valyl-tRNA synthetase (ValRS)) (10). Because the isozymes are targeted to different compartments, the two isoforms of ValRS, for example, cannot be substituted for each other in vivo. A similar scenario has been observed for genes encoding mitochondrial and cytoplasmic forms of Arabidopsis thaliana alanyl-tRNA synthetase, threonyl-tRNA synthetase, and ValRS (11).Many yeast cytoplasmic tRNA synthetases contain an N-or C-terminal polypeptide extension, which ...
BackgroundPrevious studies in Saccharomyces cerevisiae showed that ALA1 (encoding alanyl-tRNA synthetase) and GRS1 (encoding glycyl-tRNA synthetase) respectively use ACG and TTG as their alternative translation initiator codons. To explore if any other non-ATG triplets can act as initiator codons in yeast, ALA1 was used as a reporter for screening.ResultsWe show herein that except for AAG and AGG, all triplets that differ from ATG by a single nucleotide were able to serve as initiator codons in ALA1. Among these initiator codons, TTG, CTG, ACG, and ATT had ~50% initiating activities relative to that of ATG, while GTG, ATA, and ATC had ~20% initiating activities relative to that of ATG. Unexpectedly, these non-AUG initiator codons exhibited different preferences toward various sequence contexts. In particular, GTG was one of the most efficient non-ATG initiator codons, while ATA was essentially inactive in the context of GRS1.ConclusionThis finding indicates that a sequence context that is favorable for a given non-ATG initiator codon might not be as favorable for another.
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. Sequence comparison suggests that the yeast cytoplasmic (or mature mitochondrial) ValRS contains an N-terminal appendage that acts in cis as a nonspecific tRNA-binding domain (TRBD) and is absent from its bacterial relatives. We show here that Escherichia coli ValRS can substitute for the mitochondrial and cytoplasmic functions of VAS1 by fusion of a mitochondrial targeting signal and a TRBD, respectively. In addition, the bacterial ValRS gene can be converted into a dual functional yeast gene encoding both cytoplasmic and mitochondrial activities by fusion of a DNA sequence specifying both the mitochondrial targeting signal and TRBD. In vitro assays suggested that fusion of a nonspecific TRBD to the bacterial enzyme significantly enhanced its yeast tRNA-binding and aminoacylation activities. These results not only underscore the necessity of retaining a TRBD for functioning of a tRNA synthetase in yeast cytoplasm, but also provide insights into the evolution of tRNA synthetase genes.Aminoacyl-tRNA synthetases (aaRSs) 2 are a group of ancient enzymes responsible for protein translation, each of which catalyzes the attachment of a specific amino acid to its cognate tRNAs, forming aminoacyl-tRNAs. These charged tRNAs are then delivered by elongation factor-1 to ribosomes for protein translation. In prokaryotes, there are typically 20 aminoacyl-tRNA synthetases, one for each amino acid (1-4). In eukaryotes, protein synthesis occurs not only in the cytoplasm, but also in organelles, such as mitochondria and chloroplasts (5). Thus, eukaryotes, such as yeast, commonly have two genes that encode distinct sets of proteins for each aminoacylation activity, one localized in the cytoplasm and the other in the mitochondria. Each set aminoacylates the isoaccepting tRNAs within its respective cell compartment and is sequestered from the isoacceptors confined in other compartments. In most cases, cytoplasmic and mitochondrial synthetase activities are encoded by two distinct nuclear genes,
Previous studies showed that VAS1 of Saccharomyces cerevisiae encodes both cytosolic and mitochondrial forms of valyl-tRNA synthetase (ValRS) through alternative initiation of translation. We show herein that except for Schizosaccharomyces pombe, all yeast species studied contained a single ValRS gene encoding both forms, and all of the mature protein forms deduced from those genes possessed an N-terminal appended domain (Ad) that was absent from their bacterial relatives. In contrast, S. pombe contained two distinct nuclear ValRS genes, one encoding the mitochondrial form and the other its cytosolic counterpart. Although the cytosolic form closely resembles other yeast ValRS sequences (approximately 60% identity), the mitochondrial form exhibits significant divergence from others (approximately 35% identity). Both genes are active and essential for the survival of the yeast. Most conspicuously, the mitochondrial form lacks the characteristic Ad. A phylogenetic analysis further suggested that both forms of S. pombe ValRS are of mitochondrial origin, and the mitochondrial form is ancestral to the cytoplasmic form.
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