Chia-Pei Chang

Promoting the formation of an active synthetase/tRNA complex by a nonspecific tRNA-binding domain.

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 (Kd ∼ 0.5 μm). Deletion of the appended domain from the yeast enzyme severely impaired its tRNA binding, aminoacylation, and complementation activities. This N-domain-deleted yeast valyl-tRNA 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.

A single sequence context cannot satisfy all non-AUG initiator codons in yeast†

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.

Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme

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.

Alanyl-tRNA synthetase genes of Vanderwaltozyma polyspora arose from duplication of a dual-functional predecessor of mitochondrial origin

In eukaryotes, the cytoplasmic and mitochondrial forms of a given aminoacyl-tRNA synthetase (aaRS) are typically encoded by two orthologous nuclear genes, one of eukaryotic origin and the other of mitochondrial origin. We herein report a novel scenario of aaRS evolution in yeast. While all other yeast species studied possess a single nuclear gene encoding both forms of alanyl-tRNA synthetase (AlaRS), Vanderwaltozyma polyspora, a yeast species descended from the same whole-genome duplication event as Saccharomyces cerevisiae, contains two distinct nuclear AlaRS genes, one specifying the cytoplasmic form and the other its mitochondrial counterpart. The protein sequences of these two isoforms are very similar to each other. The isoforms are actively expressed in vivo and are exclusively localized in their respective cellular compartments. Despite the presence of a promising AUG initiator candidate, the gene encoding the mitochondrial form is actually initiated from upstream non-AUG codons. A phylogenetic analysis further revealed that all yeast AlaRS genes, including those in V. polyspora, are of mitochondrial origin. These findings underscore the possibility that contemporary AlaRS genes in V. polyspora arose relatively recently from duplication of a dual-functional predecessor of mitochondrial origin.

Schizosaccharomyces pombe Possesses Two Paralogous Valyl-tRNA Synthetase Genes of Mitochondrial Origin

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.

A tryptophan-rich peptide acts as a transcription activation domain.

BackgroundEukaryotic transcription activators normally consist of a sequence-specific DNA-binding domain (DBD) and a transcription activation domain (AD). While many sequence patterns and motifs have been defined for DBDs, ADs do not share easily recognizable motifs or structures.ResultsWe report herein that the N-terminal domain of yeast valyl-tRNA synthetase can function as an AD when fused to a DNA-binding protein, LexA, and turn on reporter genes with distinct LexA-responsive promoters. The transcriptional activity was mainly attributed to a five-residue peptide, WYDWW, near the C-terminus of the N domain. Remarkably, the pentapeptide per se retained much of the transcriptional activity. Mutations which substituted tryptophan residues for both of the non-tryptophan residues in the pentapeptide (resulting in W5) significantly enhanced its activity (~1.8-fold), while mutations which substituted aromatic residues with alanine residues severely impaired its activity. Accordingly, a much more active peptide, pentatryptophan (W7), was produced, which elicited ~3-fold higher activity than that of the native pentapeptide and the N domain. Further study indicated that W7 mediates transcription activation through interacting with the general transcription factor, TFIIB.ConclusionsSince W7 shares no sequence homology or features with any known transcription activators, it may represent a novel class of AD.

An Insertion Peptide in Yeast Glycyl-tRNA Synthetase Facilitates both Productive Docking and Catalysis of Cognate tRNAs

ABSTRACT The yeast Saccharomyces cerevisiae possesses two distinct glycyl-tRNA synthetase (GlyRS) genes: GRS1 and GRS2. GRS1 is dually functional, encoding both cytoplasmic and mitochondrial activities, while GRS2 is dysfunctional and not required for growth. The protein products of these two genes, GlyRS1 and GlyRS2, are much alike but are distinguished by an insertion peptide of GlyRS1, which is absent from GlyRS2 and other eukaryotic homologues. We show that deletion or mutation of the insertion peptide modestly impaired the enzymes catalytic efficiency in vitro (with a 2- to 3-fold increase in Km and a 5- to 8-fold decrease in kcat). Consistently, GRS2 can be conveniently converted to a functional gene via codon optimization, and the insertion peptide is dispensable for protein stability and the rescue activity of GRS1 at 30°C in vivo. A phylogenetic analysis further showed that GRS1 and GRS2 are paralogues that arose from a gene duplication event relatively recently, with GRS1 being the predecessor. These results indicate that GlyRS2 is an active enzyme essentially resembling the insertion peptide-deleted form of GlyRS1. Our study suggests that the insertion peptide represents a novel auxiliary domain, which facilitates both productive docking and catalysis of cognate tRNAs.

Evolutionary gain of highly divergent tRNA specificities by two isoforms of human histidyl-tRNA synthetase

The discriminator base N73 is a key identity element of tRNAHis. In eukaryotes, N73 is an “A” in cytoplasmic tRNAHis and a “C” in mitochondrial tRNAHis. 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 tRNAHis. 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 tRNAHis 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 tRNAHis, have acquired highly specialized tRNA recognition properties through evolution.

A WHEP domain regulates the dynamic structure and activity of Caenorhabditis elegans glycyl-tRNA synthetase

WHEP domains exist in certain eukaryotic aminoacyl-tRNA synthetases and play roles in tRNA or protein binding. We present evidence herein that cytoplasmic and mitochondrial forms of Caenorhabditis elegans glycyl-tRNA synthetase (CeGlyRS) are encoded by the same gene (CeGRS1) through alternative initiation of translation. The cytoplasmic form possessed an N-terminal WHEP domain, whereas its mitochondrial isoform possessed an extra N-terminal sequence consisting of an mitochondrial targeting signal and an appended domain. Cross-species complementation assays showed that CeGRS1 effectively rescued the cytoplasmic and mitochondrial defects of a yeast GRS1 knock-out strain. Although both forms of CeGlyRS efficiently charged the cytoplasmic tRNAsGly of C. elegans, the mitochondrial form was much more efficient than its cytoplasmic counterpart in charging the mitochondrial tRNAGly isoacceptor, which carries a defective TψC hairpin. Despite the WHEP domain per se lacking tRNA binding activity, deletion of this domain reduced the catalytic efficiency of the enzyme. Most interestingly, the deletion mutant possessed a higher thermal stability and a somewhat lower structural flexibility. Our study suggests a role for the WHEP domain as a regulator of the dynamic structure and activity of the enzyme.

Divergent Alanyl-tRNA Synthetase Genes of Vanderwaltozyma polyspora Descended from a Common Ancestor through Whole-Genome Duplication Followed by Asymmetric Evolution

ABSTRACT Cytoplasmic and mitochondrial forms of a eukaryotic aminoacyl-tRNA synthetase (aaRS) are generally encoded by two distinct nuclear genes, one of eukaryotic origin and the other of mitochondrial origin. However, in most known yeasts, only the mitochondrial-origin alanyl-tRNA synthetase (AlaRS) gene is retained and plays a dual-functional role. Here, we present a novel scenario of AlaRS evolution in the yeast Vanderwaltozyma polyspora. V. polyspora possesses two significantly diverged AlaRS gene homologues, one encoding the cytoplasmic form and the other its mitochondrial counterpart. Clever selection of transcription and translation initiation sites enables the two isoforms to be localized and thus functional in their respective cellular compartments. However, the two isoforms can also be stably expressed and function in the reciprocal compartments by insertion or removal of a mitochondrial targeting signal. Synteny and phylogeny analyses revealed that the AlaRS homologues of V. polyspora arose from a dual-functional common ancestor through whole-genome duplication (WGD). Moreover, the mitochondrial form had higher synonymous (1.6-fold) and nonsynonymous (2.8-fold) substitution rates than did its cytoplasmic counterpart, presumably due to a lesser constraint imposed on components of the mitochondrial translational apparatus. Our study suggests that asymmetric evolution confers the divergence between the AlaRS paralogues of V. polyspora.