Cross-species and Cross-compartmental Aminoacylation of Isoaccepting tRNAs by a Class II tRNA Synthetase
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It was previously shown that ALA1, the only alanyl-tRNA synthetase gene in Saccharomyces cerevisiae, codes for two functionally exclusive protein isoforms through alternative initiation at two consecutive ACG codons and an in-frame downstream AUG. We reported here the cloning and characterization of a homologous gene from Candida albicans. Functional assays show that this gene can substitute for both the cytoplasmic and mitochondrial functions of ALA1 in S. cerevisiae and codes for two distinct protein isoforms through alternative initiation from two in-frame AUG triplets 8-codons apart. Unexpectedly, although the short form acts exclusively in cytoplasm, the longer form provides function in both compartments. Similar observations are made in fractionation assays. Thus, the alanyl-tRNA synthetase gene of C. albicans has evolved an unusual pattern of translation initiation and protein partitioning and codes for protein isoforms that can aminoacylate isoaccepting tRNAs from a different species and from across cellular compartments. It was previously shown that ALA1, the only alanyl-tRNA synthetase gene in Saccharomyces cerevisiae, codes for two functionally exclusive protein isoforms through alternative initiation at two consecutive ACG codons and an in-frame downstream AUG. We reported here the cloning and characterization of a homologous gene from Candida albicans. Functional assays show that this gene can substitute for both the cytoplasmic and mitochondrial functions of ALA1 in S. cerevisiae and codes for two distinct protein isoforms through alternative initiation from two in-frame AUG triplets 8-codons apart. Unexpectedly, although the short form acts exclusively in cytoplasm, the longer form provides function in both compartments. Similar observations are made in fractionation assays. Thus, the alanyl-tRNA synthetase gene of C. albicans has evolved an unusual pattern of translation initiation and protein partitioning and codes for protein isoforms that can aminoacylate isoaccepting tRNAs from a different species and from across cellular compartments. Typically there are 20 aminoacyl-tRNA synthetases in prokaryotes, one for each amino acid (1Carter Jr., C.W. Annu. Rev. Biochem. 1993; 62: 715-748Crossref PubMed Scopus (327) Google Scholar, 2Martinis S.A. Schimmel P. Neidhardt F.C. Escherichia coli and Salmonella Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C1996: 887-901Google Scholar, 3Giege´ R. Sissler M. Florentz C. Nucleic Acids Res. 1998; 26: 5017-5035Crossref PubMed Scopus (633) Google Scholar, 4Pelchat M. Lapointe J. Biochem. Cell Biol. 1999; 77: 343-347Crossref PubMed Google Scholar). These enzymes each catalyze the formation of an aminoacyl-tRNA by attaching a particular amino acid to the 3′-end of its cognate tRNA, with accompanying hydrolysis of ATP to AMP and pyrophosphate. The activated amino acid, i.e. aminoacyl-tRNA, is then transferred to ribosome for protein synthesis. In eukaryotes protein synthesis occurs not only in the cytoplasm but also in organelles, such as mitochondria and chloroplasts (5Mare´chal-Drouard L. Weil J.H. Dietrich A. Annu. Rev. Cell Biol. 1993; 8: 115-131Google Scholar). Compartmentalization of the protein synthesis machinery within the cytoplasm and organelles of eukaryotes leads to isoaccepting tRNA species that are distinguished by nucleotide sequence, subcellular location, and enzyme specificity. 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. Except for some algae (6Steinmetz A. Weil J.H. Methods Enzymol. 1986; 118: 212-231Crossref Scopus (28) Google Scholar), all aminoacyl-tRNA synthetases are encoded by nuclear genes regardless of the cell compartments to which they are confined. In contrast to most known eukaryotic tRNA synthetases, two Saccharomyces cerevisiae genes, HTS1 (the gene encoding his-tidyl-tRNA synthetase) (7Natsoulis G. Hilger F. Fink G.R. Cell. 1986; 46: 235-243Abstract Full Text PDF PubMed Scopus (196) Google Scholar) and VAS1 (the gene encoding valyl-tRNA synthetase (ValRS)) (8Chatton B. Walter P. Ebel J.-P. Lacroute F. Fasiolo F. J. Biol. Chem. 1988; 263: 52-57Abstract Full Text PDF PubMed Google Scholar), specify both the mitochondrial and cytosolic forms through alternative initiation from two inframe AUG codons. Each of these genes encodes mRNAs with distinct 5′-ends. Some of these mRNAs have their 5′-ends located upstream of the first AUG codon, whereas others have their 5′-ends located between the first and second AUG codons. The mitochondrial form of the enzyme is translated from the first AUG on the “long” messages, whereas the cytosolic form is translated from the second AUG on the “short” messages. As a consequence, the mitochondrial enzymes have the same polypeptide sequences as their cytosolic counterparts, except for a short amino-terminal mitochondrial targeting sequence. The transit peptide is subsequently cleaved away upon import into the mitochondria. Because the two isoforms are targeted to different subcellular compartments, they cannot substitute for each other in vivo (7Natsoulis G. Hilger F. Fink G.R. Cell. 1986; 46: 235-243Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 8Chatton B. Walter P. Ebel J.-P. Lacroute F. Fasiolo F. J. Biol. Chem. 1988; 263: 52-57Abstract Full Text PDF PubMed Google Scholar). A similar scenario has been observed for the genes that encode the mitochondrial and cytoplasmic forms of Arabidopsis thaliana alanyl-tRNA synthetase (AlaRS), 3The abbreviations used are: AlaRS, alanyl-tRNA synthetase; ADH, alcohol dehydrogenase; 5-FOA, 5-fluoroorotic acid; ValRS, valyl-tRNA synthetase; YPG, yeast extract-peptone-glycerol; RACE, rapid amplification of cDNA ends; RT, reverse transcription. 3The abbreviations used are: AlaRS, alanyl-tRNA synthetase; ADH, alcohol dehydrogenase; 5-FOA, 5-fluoroorotic acid; ValRS, valyl-tRNA synthetase; YPG, yeast extract-peptone-glycerol; RACE, rapid amplification of cDNA ends; RT, reverse transcription. threonyl-tRNA synthetase, and ValRS (9Souciet G. Menand B. Ovesna J. Cosset A. Dietrich A. Wintz H. Eur. J. Biochem. 1999; 266: 848-854Crossref PubMed Scopus (55) Google Scholar). Recently it was shown that ALA1, the only gene coding for AlaRS in S. cerevisiae, also encodes distinct protein isoforms (10Tang H.L. Yeh L.S. Chen N.K. Ripmaster T. Schimmel P. Wang C.C. J. Biol. Chem. 2004; 279: 49656-49663Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11Chang K.J. Lin G. Men L.C. Wang C.C. J. Biol. Chem. 2006; 281: 7775-7783Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Although the cytoplasmic form is initiated from a canonical AUG triplet, its mitochondrial counterpart is initiated from two successive in-frame ACG triplets that are located 23 codons upstream of the AUG initiator, i.e. ACG(–25)/ACG(–24). These two forms function exclusively in their respective compartments and, thus, cannot substitute for each other under normal conditions. A similar scenario has been observed in GRS1 (12Chang K.J. Wang C.C. J. Biol. Chem. 2004; 279: 13778-13785Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), the only active yeast gene coding for glycyl-tRNA synthetase. Because to date examples of native non-AUG initiation are still rare in low eukaryotes (10Tang H.L. Yeh L.S. Chen N.K. Ripmaster T. Schimmel P. Wang C.C. J. Biol. Chem. 2004; 279: 49656-49663Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11Chang K.J. Lin G. Men L.C. Wang C.C. J. Biol. Chem. 2006; 281: 7775-7783Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 12Chang K.J. Wang C.C. J. Biol. Chem. 2004; 279: 13778-13785Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), we wondered whether a similar mechanism of translation initiation has been conserved in the AlaRS genes of other yeasts during evolution. In addition, we wondered whether the AlaRS gene of a closely related yeast species, such as Candida albicans, also provides function in both compartments and whether it can surmount the species barrier and charge the tRNAs of S. cerevisiae. It is our hope that results obtained from this study could provide further insight into the bifunctional nature of a particular nuclear gene and the diversity of mechanisms by which protein isoforms can be partitioned between two distinct compartments. In the work described here we presented experimental evidence that an ALA1 homologue of C. albicans (designated here as CaALA1) can rescue both the cytoplasmic and mitochondrial defects of a S. cerevisiae ala1– strain. Similar to the ALA1 gene in S. cerevisiae, two protein isoforms with distinct amino termini are alternatively generated from this gene; however, no non-canonical initiators are involved in this case. Instead, these isoforms are initiated from two in-frame AUG triplets. Even more unexpectedly, whereas the short form that is initiated from the second AUG is confined in the cytoplasm, the longer form that is initiated from the first AUG is dual-targeted and, thus, bifunctional. The implications of these observations will be further discussed in the context of co-evolution of tRNAs and their cognate tRNA synthetases. Construction of Plasmids—Cloning of CaALA1 from C. albicans followed standard protocols (13Wang W. Malcolm B.A. Biotechniques. 1999; 26: 680-682Crossref PubMed Scopus (490) Google Scholar). The wild-type CaALA1 sequence (base pairs –300∼+2910 relative to ATG1) was amplified by PCR and cloned into pRS315 (a low copy number yeast vector) or pRS425 (a high copy number yeast vector). A short sequence coding for a His6 tag or FLAG was subsequently inserted in-frame into the 3′-end of the CaALA1 open reading frame. Various point mutations, such as ATG1/ATA2 to TCT/AGA and ATG9 to GCG, were introduced into the wild-type clone following standard protocols (the number 1 in ATG1 refers to the codon position in the open reading frame). To clone CaALA1 in pADH (a high copy number yeast vector with an ADH promoter), a segment of CaALA1 DNA containing base pairs –40∼+2910 relative to ATG1 was amplified by PCR as an EagI/XhoI fragment and cloned into appropriate sites of this vector. Cloning of CaALA1-VAS1c constructs followed a strategy described earlier (12Chang K.J. Wang C.C. J. Biol. Chem. 2004; 279: 13778-13785Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Basically, various CaALA1 sequences (–370∼+102 bp, –370∼+126 bp, or –370∼+159 bp) were PCR-amplified as EagI-SpeI fragments and fused in-frame to the EagI/SpeI sites 5′ to VAS1c (the open reading frame coding for the cytoplasmic form of ValRS) cloned in a low copy number vector pRS315, resulting in various CaALA1-VAS1c constructs in which the ATG initiator for the cytoplasmic form of ValRS had been mutated. Mapping the 5′-Ends of CaALA1 Transcripts—Identification of the 5′-ends of CaALA1 transcripts was carried out with 5′-RACE (rapid amplification of cDNA ends; Invitrogen). Briefly, total RNA isolated from C. albicans was first treated with alkaline phosphatase to remove the 5′-phosphate group from truncated mRNA and non-mRNA and then with tobacco acid pyrophosphatase to remove the 5′-cap from intact full-length mRNA. An RNA oligonucleotide was subsequently fused to the 5′-end of the decapped mRNA with RNA ligase. The 5′-end modified mRNA was transcribed with SuperScript III reverse transcriptase into first strand cDNAs using an “antisense” CaALA1-specific primer that was annealed to a region 630-bp downstream of ATG1. The reaction mixture was treated with RNase H, and the first strand cDNA products were then amplified via PCR using Pfu DNA polymerase with a primer (provided by the manufacturer) annealed to the 5′-end of the cDNA and a nested CaALA1-specific primer annealed 600 bp downstream of ATG1. After PCR-driven amplification, the double-stranded cDNA products were cloned and sequenced. Sequencing of the Mitochondrial Form of CaAlaRS—Determination of the amino terminus of the processed mitochondrial form of CaAlaRS was carried out by the Edman degradation method. First, mitochondria were isolated from transformants carrying the wild-type (pIVY97) and ATG9 mutant (pIVY118) constructs (14Daum G. Bohni P.C. Schatz G. J. Biol. Chem. 1982; 257: 13028-13033Abstract Full Text PDF PubMed Google Scholar), and the His6-tagged proteins expressed were purified by nickel-nitrilotriacetic acid column chromatography. After SDS-polyacrylamide gel electrophoresis, the proteins were transferred to a nitrocellulose membrane and stained with Amido Black, and the protein band of the correct size was removed and sequenced. Complementation Assays for the Cytoplasmic Function of ALA1—The yeast ALA1 knock-out strain TRY11 was as described (15Ripmaster T.L. Shiba K. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4932-4936Crossref PubMed Scopus (40) Google Scholar). This strain is maintained by a plasmid encoding AlaRS and the URA3 marker. Complementation assays for the cytoplasmic function of plasmid-borne ALA1 and derivatives were carried out by introducing a test plasmid into TRY11 and determining the ability of transformants to grow in the presence of 5-fluoroorotic acid (5-FOA). The cultures were incubated at 30 °C for 3–5 days or until colonies appeared (photos for the complementation assays were taken at day 3 after incubation). The transformants evicted the maintenance plasmid with the URA3 marker in the presence of 5-FOA. Thus, only an enzyme with the cytoplasmic AlaRS activity encoded by the second plasmid (with the LEU2 marker) could rescue the growth defect. Complementation Assays for the Mitochondrial Function of ALA1—Complementation assays for the mitochondrial function of plasmid-borne ALA1 and derivatives were carried out by introducing a test plasmid (carrying a LEU2 marker) and a second maintenance plasmid (carrying a HIS3 marker) into TRY11 and selecting on a plate containing 5-FOA. The second maintenance plasmid used in this assay contained ALA1(I(–1)stop), which expresses a functional cytoplasmic AlaRS but is defective in mitochondrial AlaRS activity. In the presence of 5-FOA, the first maintenance plasmid (containing a URA3 marker) was evicted from the co-transformants, whereas the second maintenance plasmid was retained. Thus, all co-transformants survived 5-FOA selections due to the presence of the cytoplasmic AlaRS derived from the second maintenance plasmid. The co-transformants were further tested on YPG plates for their mitochondrial phenotypes at 30 °C, with results documented at day 3 after plating. Because a yeast cell cannot survive on glycerol without functional mitochondria, the co-transformants do not grow on YPG plates unless a functional mitochondrial AlaRS is present. Complementation assays for various VAS1 constructs and their derivatives were conducted essentially the same way as those for ALA1 constructs, except that a VAS1 knock-out strain, CW1, was used as the test strain (16Wang C.C. Chang K.J. Tang H.L. Hsieh C.J. Schimmel P. Biochemistry. 2003; 42: 1646-1651Crossref PubMed Scopus (25) Google Scholar). Western Blot—The protein expression patterns of various CaALA1 constructs were determined by a chemiluminescence-based Western blot analysis following standard protocols. The CaALA1 constructs were first transformed into INVSc1 (Novagen), and the resultant transformants were subsequently grown in a selection medium lacking leucine. The total, cytoplasmic, and mitochondrial fractions were prepared from each of the transformants according to the protocols described by Daum et al. (14Daum G. Bohni P.C. Schatz G. J. Biol. Chem. 1982; 257: 13028-13033Abstract Full Text PDF PubMed Google Scholar). 45 μg of the protein extracts were loaded onto a gel (size, 8 × 10 cm) containing 8% polyacrylamide and electrophoresed at 130 volts for 1∼2 h. The resolved proteins were transferred onto a nitrocellulose membrane using a semidry blotting device. The membrane was hybridized with a horseradish peroxidase-conjugated anti-His6 tag (Invitrogen) or anti-FLAG antibody (Sigma) and then exposed to x-ray film after the addition of the appropriate substrates. Growth Curve Assay—Growth curve assays for the plasmid-borne mitochondrial AlaRS activities were carried out in YPG broth. TRY11 was first co-transformed with a second maintenance plasmid (carrying a HIS3 marker) and a test plasmid (carrying a LEU2 marker), and the resultant co-transformants were plated on a FOA plate. After FOA selections, one colony of the survivors was picked and inoculated into 3 ml of SD broth lacking histidine and leucine and grown to stationary phase. The cells were washed three times with YPG broth, and appropriate amounts were transferred to a flask containing 10 ml of YPG broth to a final cell density of A600 = 0.1. The cell culture was shaken in a 30 °C incubator, and the cell density of the culture was checked every 4 h for a period of 48 h. Cross-species Complementation of an S. cerevisiae ala1– Strain by a Homologue of C. albicans—Unlike most yeast tRNA synthetases that have two distinct nuclear genes (one coding for the cytoplasmic enzyme and the other for its mitochondrial counterpart), CaALA1 appears to be the only ALA1 homologue in the yeast C. albicans. We wondered whether this gene actually provides AlaRS function in vivo and whether it could code for both cytoplasmic and mitochondrial activities, as with the case of ALA1 in S. cerevisiae. To further our understanding on this gene, we first scanned the 5′-terminal nucleotide sequences of this gene for potential translation start codons that might be involved in the synthesis of protein isoforms. As shown in Fig. 1A, there are two nearby in-frame ATG codons, i.e. ATG1 and ATG9, close to the 5′-end of its open reading frame. In addition, four potential non-ATG initiators, i.e. non-ATG codons that differ from ATG by a single nucleotide, are present in the sequence between ATG1 and TGA(-47), the closest termination codon. We wondered which of these triplets is the authentic start sites for CaALA1. Before proceeding, the transcription profiles of this gene in vivo were elucidated using 5′-RACE. Fig. 1B showed that a single transcript, with its 5′-end mapped to nucleotide position –24 relative to ATG1, was amplified by RT-PCR using total RNA extracts of C. albicans as the templates (Fig. 1B). This transcript was, thus, considered to be the template for protein translation. In addition, sequences of the cDNAs (∼600 bp determined) obtained from RT-PCR were identical to those of the genomic DNA (data not shown), suggesting that the possibilities of alternative splicing at the 5′-end of CaALA1 mRNAs and translation initiation from an AUG codon spliced from afar could be ruled out. Comparison of the protein sequences among AlaRSs of different origins showed that CaAlaRS (deduced from its putative open reading frame starting at ATG1) shares a significantly higher sequence identity to those from S. cerevisiae (cytoplasmic form) (∼68%) and Schizosaccharomyces pombe (∼61%) than to the Escherichia coli enzyme (∼39%) (Fig. 1C). Most intriguingly, this protein appears to have an amino-terminal 15-residue appendage that is absent from the other yeast cytoplasmic enzymes compared (Fig. 1D). It should be noted that the cytoplasmic form of AlaRS of S. cerevisiae starts from the second Met on the sequence shown in Fig. 1D. We surmised that if this protein does have mitochondrial function, this appendage could serve as at least part of its mitochondrial targeting signal. Because the genetic system has not been well developed for C. albicans, we wondered whether we could test the biological functions of CaALA1 in a closely related and well developed yeast system such as S. cerevisiae. To this end, CaALA1 cloned in various vectors was transformed into a S. cerevisiae ala1– yeast strain, TRY11, and tested for its complementing activity. As shown in Fig. 2, the wild-type CaALA1 gene cloned in pRS315 (a low copy number vector), pRS425 (a high copy number vector), or pADH (a high copy number vector with a constitutive ADH promoter) efficiently rescued both the cytoplasmic and mitochondrial defects of TRY11, i.e. transformants carrying the plasmid-borne CaALA1 gene formed colonies after 2∼3 days of growth on FOA (Fig. 2B) and YPG (Fig. 2C) plates, respectively. These results indicated that the homologous gene from C. albicans could overcome the species barrier and encodes both cytoplasmic and mitochondrial AlaRS activities. Generation of Two Functionally Overlapping Protein Isoforms through Alternative Translation Initiation—The question arose as to how many protein isoforms are generated from CaALA1 and whether the upstream non-ATG triplets (relative to ATG1) are involved in the translation initiation. To shed light on this matter, various CaALA1 constructs were cloned using pRS425 as the vector and tested for their complementation activities. First, the codon at position –1 was mutated to a stop codon TAA to block all the possible translational events initiated upstream of ATG1 and then tested for its effect on the cytoplasmic and mitochondrial functions of this gene. Fig. 3 shows that the newly introduced stop codon affected neither the mitochondrial nor cytoplasmic function of this gene (see pIVY102), suggesting that the upstream potential non-ATG initiators are not involved in the synthesis of the alanine enzyme(s). We next aimed at the two nearby ATG triplets, i.e. ATG1 and ATG9, for their possible participation in translation. Mutation of ATG1 (see pIVY100 or pSAM35) or insertion of two nucleotides between ATG1 and ATG9 (causing ATG1 to be out-of-frame with respect to the rest of the open reading frame) (see pSAM25) specifically impaired the mitochondrial function of this gene (Fig. 3, B and C), suggesting that ATG1 was the sole initiator responsible for the translation of the mitochondrial form and the cytoplasmic form was initiated elsewhere, possibly from ATG9. Further mutation of ATG9 in pIVY100 (resulting in pIVY104) abolished the remaining cytoplasmic activity, indicating that the cytoplasmic function of pIVY100 was indeed provided by protein product initiated at ATG9. However, much to our surprise, mutation of ATG9 alone did not impair the cytoplasmic function as expected (see pSAM33); instead, the ATG9 mutant still retained both activities. These results suggested that the long form that is initiated from ATG1 provided both the cytoplasmic and mitochondrial functions, whereas the shorter form that is initiated from ATG9 provided only the cytoplasmic activity. Given that the out-of-frame mutant (see pSAM25) still retained the cytoplasmic function, it is likely that the second ATG triplet can be recognized by scanning ribosomes as a remedial translation start site even in the presence of the first ATG triplet. Therefore, the cytoplasmic function of this gene is probably contributed by both isoforms under normal conditions. It is noteworthy that mutation of ATG1 to GCG (resulting in pSAM35) also created an out-of-frame ATG triplet (between the nucleotides –2 and +1) and had a phenotype similar to that of pSAM25. To provide a more quantitative data on the mitochondrial complementation activity of these mutants, a growth curve assay was subsequently carried out in YPG broth. Consistent with the observations made from the complementation assay (Fig. 3C), transformants carrying pSAM33 (with ATG9 inactivated) or pSAM20 (with the wild-type gene) grew well in YPG broth, whereas growth of those carrying pIVY100 (with ATG1 inactivated), pSAM25 (with an out-of-frame mutation between the two ATG codons), or pIVY104 (with both ATG triplets inactivated) was severely impaired (Fig. 3D). Analysis of the relative levels of specific mRNAs generated from each of these constructs indicated that similar levels of CaALA1 transcripts were generated from or maintained in the transformants carrying the wild-type or mutant CaALA1 constructs as determined by a semiquantitative RT-PCR experiment (Fig. 3E). This observation suggested that these mutations had little effect on the stability of the specific mRNAs generated from the constructs and, therefore, most likely modulated only the translation initiation activity of the individual initiators. Because the observations made above were a result of AlaRS proteins produced from a high copy number plasmid, we were afraid that they might not accurately reflect protein activity at physiological levels. Therefore, wild-type and mutant CaALA1 genes were cloned into a low copy number shuttle vector (Fig. 4A) and tested for their cytoplasmic and mitochondrial functions. As shown in Fig. 4, B and C, these constructs had complementing activities similar to the corresponding constructs cloned in a high copy number vector. Partition Pattern of ATG1- and ATG9-initiated CaAlaRS Isoforms—To investigate whether the CaALA1 constructs contain similar activities when highly expressed from a constitutive ADH promoter, some of the representative constructs shown in Fig. 3A were subcloned into pADH and tested for their complementation functions. As shown in Fig. 5, A–C, these constructs contained similar complementation functions to those cloned in pRS425, except for the ATG1 mutant, which contained only cytoplasmic function when cloned in pRS425 but contained both cytoplasmic and mitochondrial functions when cloned in pADH (compare pIVY100 and pIVY98). One likely possibility leading to this outcome is that the ATG9-initiated form contains a cryptic mitochondrial targeting signal that normally does not play a role in mitochondrial localization but can be recruited to function when the protein is highly expressed. To directly look at the protein expression levels and elucidate the correlations between complementation functions and partition patterns of the isoforms within the cell, the total, mitochondrial, and cytoplasmic fractions were isolated from each of the transformants harboring various CaALA1 constructs. As shown in Fig. 5D, the proteins expressed from the wild-type CaALA1 construct were partitioned between cytoplasm and mitochondria (lane 1, pIVY97), but mutations that inactivated both of the ATG initiators completely abolished the synthesis of the isoforms (lane 4, pIVY149). Interestingly, when the first ATG initiator was inactivated (lane 2, pIVY98), the protein levels in the total fraction remained almost unchanged, whereas the protein band in the mitochondrial fraction drastically decreased (compare lanes 1 and 2), suggesting that the mitochondrial proteins came largely from initiation at ATG1. In addition, it came as a surprise to us at first to find that the protein levels in the cytoplasmic fraction of this mutant appreciably increased. We surmised that the basis underlying this unexpected observation could probably be attributed to the fact that ATG9 served only as a remedial initiation site in the wild-type construct (lane 1) but became the first available ATG initiator in the ATG1 mutant (lane 2), leading to the higher expression of the “cytoplasmic” form. To assess the initiating activity of the remedial initiation site, i.e. ATG9, more accurately, ATG1 was left unaltered, and two extra nucleotides were inserted into the sequence between the two ATG initiators, causing the first ATG to be out-of-frame with respect to the rest of the open reading frame (lane 5, pIVY152). Under such conditions the protein band in the total fraction only slightly decreased due to loss of the ATG1-initiated protein, whereas the protein level in the cytoplasmic fraction was almost unchanged (compare lanes 1 and 5). But most significant of all, no protein was seen in the mitochondrial fraction of this out-of-frame mutant (lane 5), suggesting that the ATG9-initiated protein form was exclusively confined to the cytoplasm when ATG9 serves only as a remedial initiation site (compare lanes 1 and 5) and could be forced into mitochondria, possibly due to the presence of a cryptic mitochondrial targeting signal, when ATG9 serves as the first available initiator, resulting in higher expression of the short form (compare lanes 2 and 5). By contrast, when the second ATG initiator was inactivated (lane 3; pIVY118), the levels of the proteins in the total or cytoplasmic extracts drastically decreased, suggesting that the cytoplasmic proteins came largely from initiation at ATG9 in the wild-type construct (compare lanes 1 and 3), and the ATG1-initiated protein form can be partitioned in both compartments (lane 3), with the major portion targeted to the mitochondria. It is noteworthy that the protein level in the mitochondrial fraction of this mutant appreciably increased as compared with the wild-type construct (compare lanes 1 and 3). We surmised that perhaps this unexpected increase was due to alterations of the potential MPP cleavage site, which might affect its processing and in turn its distribution. As a control, the mitochondrial and cytoplasmic fractions were also probed with a mixture of antiphosphoglycerate kinase (a cytoplasmic marker protein) and anti-Hsp60 (a mitochondrial marker protein) to check for cross-contamination. As shown in Fig. 5D, no serious cross-contamination was seen in these preparations (lower two panels). To further quantify the initiating activity of ATG1 and ATG9, the relative protein levels in the total fractions of pIVY152 (an out-of-frame mutant) and pIVY118 (an ATG9 mutant) were compared. Fig. 5E showed that the initiating activity of ATG9 is around 4-fold as high as that of ATG1 under the conditions used. As a control, the relative protein levels generated from pIVY97 (wild type) were also shown. This result suggKeywords:
Aminoacylation
Amino Acyl-tRNA Synthetases
Amino Acyl-tRNA Synthetases
Amino Acyl-tRNA Synthetases
Aminoacylation
Aminoacyl-tRNA
Moiety
Amino Acyl-tRNA Synthetases
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The correct aminoacylation of tRNA with the proper aminoacid by aminoacyl-tRNA synthetase is one of the key reactions which determines the overall high fidelity of protein biosynthesis. The initial selection of the amino acid is achieved in the active centre of the synthetase at the activation step due to differences in the side chains binding energies of specific substrate and the competing amino acids present in cell. If, nevertheless, the activation of amino acids structurally similar to the cognate one does proceed, additional mechanisms of correction which are based on the decomposition of unstable noncognate (intermediate or final) product of the tRNA aminoacylation reaction, by synthetase are switched on. In this review the literature on the specificity of aminoacyl-tRNA synthetases at amino acid activation step is analyzed along with the proofreading mechanisms which allow the elimination of the errors, leading to so called superspecifity of aminoacyl-tRNA synthetases.
Aminoacylation
Amino Acyl-tRNA Synthetases
Proofreading
Amino Acyl-tRNA Synthetases
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Abstract Aminoacyl‐tRNA synthetases (aaRSs) compose a family of essential enzymes that attach amino acids covalently to tRNA molecules during protein synthesis. Some aaRSs possess a hydrolytic amino acid editing function to ensure the fidelity of protein synthesis. In addition, aminoacylation can occur by indirect pathways that rely on mischarged tRNA intermediates and enzymes other than aaRSs. Throughout evolution, structural and functional divergence of aaRSs has yielded diverse secondary roles. Likewise, aaRS‐like proteins with either sequence or structural similarities to synthetases exhibit functions that may or may not be related to aminoacylation. Many of these diverse aaRSs and aaRS‐like proteins have been capitalized on by the microbial world and by medical research as targets for therapeutic agents such as antibiotics.
Amino Acyl-tRNA Synthetases
Aminoacylation
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Accuracy of aminoacylation is dependent on maintaining fidelity during attachment of amino acids to cognate tRNAs. Cis- and trans-editing protein factors impose quality control during protein translation and 8 of 36 Plasmodium falciparum aminoacyl-tRNA synthetase (aaRS) assemblies contain canonical putative editing modules. Based on expression and localization profiles of these 8 aaRSs, we propose an asymmetric distribution between the parasite cytoplasm and its apicoplast of putative editing-domain containing aaRSs. We also show that the single copy alanyl- and threonyl-tRNA synthetases are dually targeted to parasite cytoplasm and apicoplast. This bipolar presence of two unique synthetases presents opportunity for inhibitor targeting their aminoacylation and editing activities in twin parasite compartments. We used this approach to identify specific inhibitors against the alanyl- and threonyl-tRNA synthetases. Further development of such inhibitors may lead to anti-parasitics which simultaneously block protein translation in two key parasite organelles, a strategy of wider applicability for pathogen control.
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Aminoacylation
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The accuracy of protein biosynthesis rests on the high fidelity with which aminoacyl-tRNA synthetases discriminate between tRNAs. Correct aminoacylation depends not only on identity elements (nucleotides in certain positions) in tRNA (1), but also on competition between different synthetases for a given tRNA (2). Here we describe in vivo and in vitro experiments which demonstrate how variations in the levels of synthetases and tRNA affect the accuracy of aminoacylation. We show in vivo that concurrent overexpression of Escherichia coli tyrosyl-tRNA synthetase abolishes misacylation of supF tRNA Tyr with glutamine in vivo by overproduced glutaminyl-tRNA synthetase. In an in vitro competition assay, we have confirmed that the overproduction mischarging phenomenon observed in vivo is due to competition between the synthetases at the level of aminoacylation. Likewise, we have been able to examine the role competition plays in the identity of a non-suppressor tRNA of ambiguous identity, tRNA Glu . Finally, with this assay, we show that the identity of a tRNA and the accuracy with which it is recognized depend on the relative affinities of the synthetases for the tRNA. The in vitro competition assay represents a general method of obtaining qualitative information on tRNA identity in a competitive environment (usually only found in vivo ) during a defined step in protein biosynthesis, aminoacylation. In addition, we show that the discriminator base (position 73) and the first base of the anticodon are important for recognition by E. coli tyrosyl-tRNA synthetase.
Aminoacylation
Amino Acyl-tRNA Synthetases
T arm
Amino Acyl-tRNA Synthetases
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Aminoacylation
Amino Acyl-tRNA Synthetases
Amino Acyl-tRNA Synthetases
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Citations (48)
The algorithm of the genetic code is a “rosetta stone” that connects the RNA World to the theatre of proteins (Fig. 1). That connection comes from the aminoacylation of RNA. The ester linkage of an aminoacyl-RNA is higher in energy than that of the peptide bond. When two aminoacyl groups are brought into close proximity, spontaneous peptide-bond formation creates a peptidyl-RNA that can continue to react with other aminoacyl-RNAs to build up a polypeptide chain linked to RNA. The aminoacylation reaction is thus at the heart of the transition from the RNA World to the theatre of proteins. For this reason, aminoacyl-tRNA synthetases (AARS) are center stage for investigations of the origins of living systems. Through catalysis of the aminoacylation reaction, these enzymes match amino acids (AA) with nucleotide triplets imbedded (as anticodons) in tRNAs. The reaction typically occurs in two steps:(1)AA+ATP+AARS→AARS(AA-AMP)+PPi(2)AARS(AA-AMP)+tRNA→AA-tRNA+AMP+AARS In the first reaction, a tightly bound aminoacyl adenylate (AA-AMP) is formed. In the second reaction, the amino acid is transferred from the bound adenylate to the tRNA to form AA-tRNA, where the amino acid is linked through an aminoacyl ester to the 3′ -end of the tRNA. It is in the second reaction (the so-called transfer reaction) that the algorithm of the code is established. Each amino acid has its own aminoacyl-tRNA synthetases, so that 20 enzymes are needed. Because of the degeneracy of the code, each amino acid has more than one tRNA isoacceptor charged by the same synthetase. The code itself...
Aminoacylation
Amino Acyl-tRNA Synthetases
Genetic Code
Amino Acyl-tRNA Synthetases
Aminoacyl-tRNA
EF-Tu
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Aminoacylation
Amino Acyl-tRNA Synthetases
Aminoacyl-tRNA
Amino Acyl-tRNA Synthetases
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Aminoacyl-tRNA synthetase (aaRS) catalyzes the esterbond formation between the cognate amino acid andtRNA(s). The aminoacylation of tRNA is carried out in atwo-step reaction, the formation of an active intermediate(aminoacyl-adenylate or aa-AMP) from the amino acidand ATP, and the transfer of the aminoacyl moiety of theaa-AMP to the 3′ terminal adenosine (A76) of the tRNA.Strict recognition of both the amino acid and the tRNA bythe aaRS ensures the correct translation of the genetic code...
Aminoacylation
Amino Acyl-tRNA Synthetases
Genetic Code
Amino Acyl-tRNA Synthetases
Moiety
Aminoacyl-tRNA
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The rules of the genetic code are determined by the specific aminoacylation of transfer RNAs by aminoacyl transfer RNA synthetases. A straightforward analysis shows that a system of synthetase-tRNA interactions that relies on anticodons for specificity could, in principle, enable most synthetases to distinguish their cognate tRNA isoacceptors from all others. Although the anticodons of some tRNAs are recognition sites for the cognate aminoacyl tRNA synthetases, for other synthetases the anticodon is dispensable for specific aminoacylation. In particular, alanine and histidine tRNA synthetases aminoacylate small RNA minihelices that reconstruct the part of their cognate tRNAs that is proximate to the amino acid attachment site. Helices with as few as six base pairs can be efficiently aminoacylated. The specificity of aminoacylation is determined by a few nucleotides and can be converted from one amino acid to another by the change of only a few nucleotides. These findings suggest that, for a subgroup of the synthetases, there is a distinct code in the acceptor helix of transfer RNAs that determines aminoacylation specificity.—Schimmel, P. RNA minihelices and the decoding of genetic information. FASEB J. 5: 2180–2187; 1991.
Aminoacylation
Genetic Code
Amino Acyl-tRNA Synthetases
Alanine
Stathmin
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