Adaptation of aminoacylation identity rules to mammalian mitochondria

Many mammalian mitochondrial aminoacyl-tRNA synthetases are of bacterial-type and share structural domains with homologous bacterial enzymes of the same specificity. Despite this high similarity, synthetases from bacteria are known for their inability to aminoacylate mitochondrial tRNAs, while mitochondrial enzymes do aminoacylate bacterial tRNAs. Here, the reasons for non-aminoacylation by a bacterial enzyme of a mitochondrial tRNA have been explored. A mutagenic analysis performed on in vitro transcribed human mitochondrial tRNA^Asp variants tested for their ability to become aspartylated by Escherichia coli aspartyl-tRNA synthetase, reveals that full conversion cannot be achieved on the basis of the currently established tRNA/synthetase recognition rules. Integration of the full set of aspartylation identity elements and stabilization of the structural tRNA scaffold by restoration of D- and T-loop interactions, enable only a partial gain in aspartylation efficiency. The sequence context and high structural instability of the mitochondrial tRNA are additional features hindering optimal adaptation of the tRNA to the bacterial enzyme. Our data support the hypothesis that non-aminoacylation of mitochondrial tRNAs by bacterial synthetases is linked to the large sequence and structural relaxation of the organelle encoded tRNAs, itself a consequence of the high rate of mitochondrial genome divergence.     

Effects of adenosine triphosphate and magnesium chloride on affinity elution of aminoacyl-transfer ribonucleic acid synthetases from phosphocellulose with transfer ribonucleic acids.

Aminoacyl-tRNA synthetases of bakers' yeast (Saccharomyces cerevisiae) were adsorbed to a phosphocellulose (P-cellulose) column, and those specific for tyrosine [EC 6.1.1.1], threonine [EC 6.1.1.3], valine [EC 6.1.1.9], and isoleucine [EC 6.1.1.5] were eluted with several specific tRNAs. Elutions of these synthetases were affected by ATP and/or MgCl2. The effects of ATP and MgCl2 differ with synthetases. Elutions of tyrosyl- and valyl-tRNA synthetases with their cognate tRNAs were more specific in the presence of MgCl2. Isoleucyl-tRNA synthetase was eluted with its cognate tRNA in the presence of both ATP and MgCl2. On the other hand, threonyl-tRNA synthetase was eluted in the absence of ATP and MgCl2 with unfractionated tRNA but not with some non-cognate tRNAs. This suggests that elution of threonyl-tRNA synthetase is highly specific. The present data on the effects of ATP or MgCl2 or both on this affinity elution will be useful for simple and rapid purification of the synthetases.     

Ancestral AlaX Editing Enzymes for Control of Genetic Code Fidelity Are Not tRNA-specific

Accurate protein synthesis requires the hydrolytic editing of tRNAs incorrectly aminoacylated by aminoacyl-tRNA synthetases (ARSs). Recognition of cognate tRNAs by ARS is less error-prone than amino acid recognition, and, consequently, editing domains are generally believed to act only on the tRNAs cognate to their related ARSs. For example, the AlaX family of editing domains, including the editing domain of alanyl-tRNA synthetase and the related free-standing trans-editing AlaX enzymes, are thought to specifically act on tRNA^Ala, whereas the editing domains of threonyl-tRNA synthetases are specific for tRNA^Thr. Here we show that, contrary to this belief, AlaX-S, the smallest of the extant AlaX enzymes, deacylates Ser-tRNA^Thr in addition to Ser-tRNA^Ala and that a single residue is important to determine this behavior. Our data indicate that promiscuous forms of AlaX are ancestral to tRNA-specific AlaXs. We propose that former AlaX domains were used to maintain translational fidelity in earlier stages of genetic code evolution when mis-serylation of several tRNAs was possible.     

Misacylation of tRNA with methionine in Saccharomyces cerevisiae

Accurate transfer RNA (tRNA) aminoacylation by aminoacyl-tRNA synthetases controls translational fidelity. Although tRNA synthetases are generally highly accurate, recent results show that the methionyl-tRNA synthetase (MetRS) is an exception. MetRS readily misacylates non-methionyl tRNAs at frequencies of up to 10% in mammalian cells; such mismethionylation may serve a beneficial role for cells to protect their own proteins against oxidative damage. The Escherichia coli MetRS mismethionylates two E. coli tRNA species in vitro, and these two tRNAs contain identity elements for mismethionylation. Here we investigate tRNA mismethionylation in Saccharomyces cerevisiae. tRNA mismethionylation occurs at a similar extent in vivo as in mammalian cells. Both cognate and mismethionylated tRNAs have similar turnover kinetics upon cycloheximide treatment. We identify specific arginine/lysine to methionine-substituted peptides in proteomic mass spectrometry, indicating that mismethionylated tRNAs are used in translation. The yeast MetRS is part of a complex containing the anchoring protein Arc1p and the glutamyl-tRNA synthetase (GluRS). The recombinant Arc1p–MetRS–GluRS complex binds and mismethionylates many tRNA species in vitro. Our results indicate that the yeast MetRS is responsible for extensive misacylation of non-methionyl tRNAs, and mismethionylation also occurs in this evolutionary branch.     

The mitochondrial and cytoplasmic valyl tRNA synthetases in Tetrahymena are indistinguishable.

The mitochondrial and cytoplasmic valyl tRNA synthetases from Tetrahymena pyriformis are indistinguishable. These synthetases cannot be differentiated through hydroxylapatite, DEAE-cellulose, or phosphocellulose column chromatography. Both enzymes show the same mean sedimentation coefficient of 5.9 S in sucrose gradient centrifugation analysis; when bound with tRNA, they are relatively stable and sediment at 7.8 S. The temperature optimum for aminoacylation reaction is 27.5 °C, the optimum Mg²⁺ concentration is 4.4 mm, and substrate affinities (K_m values) for valine and ATP in aminoacylation are the same for both enzymes at 1.0 μm and 2.5 mm, respectively. These enzymes show identical specificities for acylation of different tRNA species, i.e., Tetrahymena and rat liver tRNAs can be equally well recognized, but no significant acylation can be observed with Escherichia coli and Saccharomyces tRNAs. These observations suggest the probable molecular identity of mitochondrial and cytoplasmic valyl tRNA synthetases in Tetrahymena.     

Initial position of aminoacylation of individual Escherichia coli, yeast, and calf liver transfer RNAs.

Transfer RNAs from Escherichia coli, yeast (Sacharomyces cerevisiae), and calf liver were subjected to controlled hydrolysis with venom exonuclease to remove 3'-terminal nucleotides, and then reconstructed successively with cytosine triphosphate (CTP) and 2'- or 3'-deoxyadenosine 5'-triphosphate in the presence of yeast CTP(ATP):tRNA nucleotidyltransferase. The modified tRNAs were purified by chromatography on DBAE-cellulose or acetylated DBAE-cellulose and then utilized in tRNA aminoacylation experiments in the presence of the homologous aminoacyl-tRNA synthetase activities. The E. coli, yeast, and calf liver aminoacyl-tRNA synthetases specific for alanine, glycine, histidine, lysine, serine, and threonine, as well as the E. coli and yeast prolyl-tRNA synthetases and the yeast glutaminyl-tRNA synthetase utilized only those homologous modified tRNAs terminating in 2'-deoxyadenosine (i.e., having an available 3'-OH group). This is interpreted as evidence that these aminoacyl-tRNA synthetases normally aminoacylate their unmodified cognate tRNAs on the 3'-OH group. The aminoacyl-tRNA synthetases from all three sources specific argining, isoleucine, leucine, phenylalanine, and valine, as well as the E. coli and yeast enzymes specific for methionine and the E. coli glutamyl-tRNA synthetase, used as substrates exclusively those tRNAs terminating in 3'-deoxyadenosine. Certain aminoacyl-tRNA synthetases, including the E. coli, yeast, and calf liver asparagine and tyrosine activating enzymes, the E. coli and yeast cysteinyl-tRNA synthetases, and the aspartyl-tRNA synthetase from yeast, utilized both isomeric tRNAs as substrates, although generally not at the same rate. While the calf liver aspartyl- and cysteinyl-tRNA synthetases utilized only the corresponding modified tRNA species terminating in 2'-deoxyadenosine, the use of a more concentrated enzyme preparation might well result in aminoacylation of the isomeric species. The one tRNA for which positional specificity does seem to have changed during evolution is tryptophan, whose E. coli aminoacyl-tRNA synthetase utilized predominantly the cognate tRNA terminating in 3'-deoxyadenosine, while the corresponding yeast and calf liver enzymes were found to utilize predominantly the isomeric tRNAs terminating in 2'-deoxyadenosine. The data presented indicate that while there is considerable diversity in the initial position of aminoacylation of individual tRNA isoacceptors derived from a single source, positional specificity has generally been conserved during the evolution from a prokaryotic to mammalian organism.     

氨酰-tRNA合成酶对tRNA的识别

氨酰-tRNA合成酶(aaRS)与tRNA的相互作用保证了蛋白质生物合成的忠实性. 氨酰-tRNA合成酶对tRNA识别的专一性依赖于aaRS特定的催化结构域和tRNA分子特异的三级结构构象. 反密码子和接受茎(包括73位)在大多数aaRS对tRNA分子的识别过程中起着关键作用, 其他部位如可变口袋、可变(茎)环等, 甚至修饰核苷酸对于一些识别过程也有重要作用.     

Maintaining genetic code through adaptations of tRNA synthetases to taxonomic domains

The universal genetic code is determined by the aminoacylation of tRNAs. In spite of the universality of the code, there are barriers to aminoacylation across taxonomic domains. These barriers are thought to correlate with the co-segregation of sequences of synthetases and tRNAs into distinct taxonomic domains. By contrast, we show here examples of eukaryote-like synthetases that are found in certain prokaryotes. The associated tRNAs have retained their prokaryote-like character in each instance. Thus, co-segregation of domain-specific synthetases and tRNAs does not always occur. Instead, synthetases make adaptations of tRNA-protein contacts to cross taxonomic domains.     

The essentiality of sulfhydryl groups to transport in Neurospora crassa.

Unfractionated Escherichia coli B tRNAs have been aminoacylated with selenocysteine by using homologous aminoacyl synthetases. Cochromatography of [³H]cysteyl-tRNA and [⁷⁵Se]selenocysteyl-tRNA on reverse-phase chromatography-5 columns revealed nearly coincident radioactive elution profiles for the two charged tRNAs. Acylation of a mixture of tRNAs with cysteine protected selenocysteine-acceptor activity from inactivation by periodate oxidation. Likewise, preacylation with selenocysteine protected cysteine acceptor from oxidation. Levels of charging with cysteine are reduced about 50% by the presence of a 40-fold excess of selenocysteine. These results indicate that selenocysteine is bound to cysteine-accepting tRNAs, although it does have considerably lower affinity for the ligase than cysteine. The ester linkage of selenocysteyl-tRNA was shown to be somewhat more stable than that of cysteyl-tRNA under the same conditions. These experiments show that selenocysteine can participate in the early steps leading to peptide-bond formation and provide a possible pathway for selenocysteine incorporation into protein.     

Transfer RNA gene mapping studies on chloroplast DNA from Chlamydomonas reinhardii

Total tRNA of Chlamydomonas reinhardii was fractionated by 2-dimensional gel electrophoresis. Sixteen tRNAs specific for eleven amino acids could be identified by aminoacylation with Escherichia coli tRNA synthetases. Hybridization of these tRNAs with chloroplast restriction fragments allowed for the localization of the genes of tRNA^Tyr, tRNA^Pro, tRNA^Phe (2 genes), tRNA^Ile (2 genes) and tRNA^His (2 genes) on the chloroplast genome of C. reinhardii. The genes for tRNA^Ala (2 genes), tRNA^Asn and tRNA^Leu were mapped by using individual chloroplast tRNAs from higher plants as probes.     

Partition of tRNAGly isoacceptors between protein and cell-wall peptidoglycan synthesis in Staphylococcus aureus

The sequence of tRNAs is submitted to evolutionary constraints imposed by their multiple interactions with aminoacyl-tRNA synthetases, translation elongation factor Tu in complex with GTP (EF-Tu•GTP), and the ribosome, each being essential for accurate and effective decoding of messenger RNAs. In Staphylococcus aureus, an additional constraint is imposed by the participation of tRNA^Gly isoacceptors in the addition of a pentaglycine side chain to cell-wall peptidoglycan precursors by transferases FmhB, FemA and FemB. Three tRNA^Gly isoacceptors poorly interacting with EF-Tu•GTP and the ribosome were previously identified. Here, we show that these ‘non-proteogenic’ tRNAs are preferentially recognized by FmhB based on kinetic analyses and on synthesis of stable aminoacyl-tRNA analogues acting as inhibitors. Synthesis of chimeric tRNAs and of helices mimicking the tRNA acceptor arms revealed that this discrimination involves identity determinants exclusively present in the D and T stems and loops of non-proteogenic tRNAs, which belong to an evolutionary lineage only present in the staphylococci. EF-Tu•GTP competitively inhibited FmhB by sequestration of ‘proteogenic’ aminoacyl-tRNAs in vitro. Together, these results indicate that competition for the Gly-tRNA^Gly pool is restricted by both limited recognition of non-proteogenic tRNAs by EF-Tu•GTP and limited recognition of proteogenic tRNAs by FmhB.     

Eukaryotic tRNA sequences present conserved and amino acid-specific structural signatures

Metazoan organisms have many tRNA genes responsible for decoding amino acids. The set of all tRNA genes can be grouped in sets of common amino acids and isoacceptor tRNAs that are aminoacylated by corresponding aminoacyl-tRNA synthetases. Analysis of tRNA alignments shows that, despite the high number of tRNA genes, specific tRNA sequence motifs are highly conserved across multicellular eukaryotes. The conservation often extends throughout the isoacceptors and isodecoders with, in some cases, two sets of conserved isodecoders. This study is focused on non-Watson–Crick base pairs in the helical stems, especially GoU pairs. Each of the four helical stems may contain one or more conserved GoU pairs. Some are amino acid specific and could represent identity elements for the cognate aminoacyl tRNA synthetases. Other GoU pairs are found in more than a single amino acid and could be critical for native folding of the tRNAs. Interestingly, some GoU pairs are anticodon-specific, and others are found in phylogenetically-specific clades. Although the distribution of conservation likely reflects a balance between accommodating isotype-specific functions as well as those shared by all tRNAs essential for ribosomal translation, such conservations may indicate the existence of specialized tRNAs for specific translation targets, cellular conditions, or alternative functions.     

The characterization of the RNAs and aminoacyl-tRNA synthetases of the blue-green alga, Anacystis nidulans

The tRNA and aminoacyl-tRNA synthetases of the blue-green alga, Anacystis nidulans have been isolated and studied. The distribution of some algal tRNA species on BD-cellulose chromatography has been determined. One tRNA^Met species has been isolated in 80% purity by a single chromatography on a BD-cellulose column developed with a modified salt gradient. The number of different tRNA isoacceptors for Met, Ser, and Leu has been ascertained by RPC-5 chromatography. The recognition of algal tRNAs by the homologous algal synthetase preparation as well as the heterologous Escherichia coli preparation was studied by the aminoacylation tests. Since all of the isoaccepting species of the tRNAs tested behaved almost identically in presence of the two enzyme preparations, a conservation of the recognition site during the evolutionary divergence of bacteria and algae is strongly suggested.     

Increased expression of tryptophan and tyrosine tRNAs elevates stop codon readthrough of reporter systems in human cell lines

Regulation of translation via stop codon readthrough (SC-RT) expands not only tissue-specific but also viral proteomes in humans and, therefore, represents an important subject of study. Understanding this mechanism and all involved players is critical also from a point of view of prospective medical therapies of hereditary diseases caused by a premature termination codon. tRNAs were considered for a long time to be just passive players delivering amino acid residues according to the genetic code to ribosomes without any active regulatory roles. In contrast, our recent yeast work identified several endogenous tRNAs implicated in the regulation of SC-RT. Swiftly emerging studies of human tRNA-ome also advocate that tRNAs have unprecedented regulatory potential. Here, we developed a universal U6 promotor-based system expressing various human endogenous tRNA iso-decoders to study consequences of their increased dosage on SC-RT employing various reporter systems in vivo. This system combined with siRNA-mediated downregulations of selected aminoacyl-tRNA synthetases demonstrated that changing levels of human tryptophan and tyrosine tRNAs do modulate efficiency of SC-RT. Overall, our results suggest that tissue-to-tissue specific levels of selected near-cognate tRNAs may have a vital potential to fine-tune the final landscape of the human proteome, as well as that of its viral pathogens.     

Bovine mitochondrial tRNAPhe, tRNASer (AGY) and tRNASer (UCN): preparation using a new detection method and their properties in aminoacylation

Bovine mitochondrial tRNAPhe, tRNASer (AGY), and tRNASer (UCN) possessing unusual structures were purified using a new hybridization assay system and their properties in aminoacylation were examined. Bovine mitochondrial phenyl-alanyl- and seryl-tRNA synthetases could aminoacylate the same amino acid-specific tRNAs obtained not only from the mitochondria but also from other sources such as E. coli, Thermus thermophilus, bovine and yeast cytosols and archaebacteria, Sulfolobus acidocaldarius. On the contrary, none of both bacterial and cytosolic synthetases could aminoacylate the same amino acid specific tRNAs from the heterologous sources with some exceptions. We consider that the bovine mitochondrial aminoacyl-tRNA synthetases have considerably simple recognition mechanism toward the substrate tRNAs compared with the non-mitochondrial ones. This mechanism may be correlated with the occurrence of structural varieties of the mitochondrial tRNA species with unusual structures.     

Mutational analysis of the tRNA mimicry of brome mosaic virus RNA. Sequence and structural requirements for aminoacylation and 3'-adenylation

The genomic RNAs of brome mosaic virus (BMV) exhibit various tRNA-like properties, including specific tyrosylation by tyrosyl-tRNA synthetases and adenylation of the 3'-CCOH derivative by tRNA nucleotidyl transferases. We have studied the effect of numerous mutations in all domains of the tRNA-like structure of BMV RNA on tyrosylation and adenylation in vitro. Surprisingly few mutations resulted in more than 50% decrease in tyrosylation rates with either wheat germ or yeast synthetases; those mutations were at the 3' terminus, the pseudoknot, and the bases of arms B and E. The results suggest an interaction of synthetase with arm A as the analog of the aminoacyl acceptor stem of tRNAs, and arm B as the analog of the anticodon arm of tRNAs, although there is no apparent interaction with the terminal loop of arm B analogous to the interaction with the anticodon in tRNAs. Mutations at several loci resulted in large losses of adenylation activity catalyzed by wheat germ and Escherichia coli nucleotidyl transferases; those loci were the pseudoknot, the bases of arms B, C and D, and at the junctions of these arms with arm A. These studies have identified mutants specifically defective in one of the tRNA-like activities, which are appropriate for investigating the role of these activities during infection in vivo.     

Handling mammalian mitochondrial tRNAs and aminoacyl-tRNA synthetases for functional and structural characterization

The mammalian mitochondrial (mt) genome codes for only 13 proteins, which are essential components in the process of oxidative phosphorylation of ADP into ATP. Synthesis of these proteins relies on a proper mt translation machinery. While 22 tRNAs and 2 rRNAs are also coded by the mt genome, all other factors including the set of aminoacyl-tRNA synthetases (aaRSs) are encoded in the nucleus and imported. Investigation of mammalian mt aminoacylation systems (and mt translation in general) gains more and more interest not only in regard of evolutionary considerations but also with respect to the growing number of diseases linked to mutations in the genes of either mt-tRNAs, synthetases or other factors. Here we report on methodological approaches for biochemical, functional, and structural characterization of human/mammalian mt-tRNAs and aaRSs. Procedures for preparation of native and in vitro transcribed tRNAs are accompanied by recommendations for specific handling of tRNAs incline to structural instability and chemical fragility. Large-scale preparation of mg amounts of highly soluble recombinant synthetases is a prerequisite for structural investigations that requires particular optimizations. Successful examples leading to crystallization of four mt-aaRSs and high-resolution structures are recalled and limitations discussed. Finally, the need for and the state-of-the-art in setting up an in vitro mt translation system are emphasized. Biochemical characterization of a subset of mammalian aminoacylation systems has already revealed a number of unprecedented peculiarities of interest for the study of evolution and forensic research. Further efforts in this field will certainly be rewarded by many exciting discoveries.