Higher-order structure of bovine mitochondrial tRNA(SerUGA): chemical modification and computer modeling.

On the basis of enzymatic probing and phylogenetic comparison, we have previously proposed that mammalian mitochondrial tRNA(sSer) (anticodon UGA) possess a slightly altered cloverleaf structure in which only one nucleotide exists between the acceptor stem and D stem (usually two nucleotides) and the anticodon stem consists of six base pairs (usually five base pairs) [Yokogawa et al. (1991) Nucleic Acids Res. 19, 6101-6105]. To ascertain whether such tRNA(sSer) can be folded into a normal L-shaped tertiary structure, the higher-order structure of bovine mitochondrial tRNA(SerUGA) was examined by chemical probing using dimethylsulfate and diethylpyrocarbonate, and on the basis of the results a tertiary structure model was obtained by computer modeling. It was found that a one-base-pair elongation in the anticodon stem was compensated for by multiple-base deletions in the D and extra loop regions of the tRNA(SerUGA), which resulted in preservation of an L-shaped tertiary structure similar to that of conventional tRNAs. By summarizing the findings, the general structural requirements of mitochondrial tRNAs necessary for their functioning in the mitochondrial translation system are considered.     

Mamit-tRNA, a database of mammalian mitochondrial tRNA primary and secondary structures

Mamit-tRNA (http://mamit-tRNA.u-strasbg.fr), a database for mammalian mitochondrial genomes, has been developed for deciphering structural features of mammalian mitochondrial tRNAs and as a helpful tool in the frame of human diseases linked to point mutations in mitochondrial tRNA genes. To accommodate the rapid growing availability of fully sequenced mammalian mitochondrial genomes, Mamit-tRNA has implemented a relational database, and all annotated tRNA genes have been curated and aligned manually. System administrative tools have been integrated to improve efficiency and to allow real-time update (from GenBank Database at NCBI) of available mammalian mitochondrial genomes. More than 3000 tRNA gene sequences from 150 organisms are classified into 22 families according to the amino acid specificity as defined by the anticodon triplets and organized according to phylogeny. Each sequence is displayed linearly with color codes indicating secondary structural domains and can be converted into a printable two-dimensional (2D) cloverleaf structure. Consensus and typical 2D structures can be extracted for any combination of primary sequences within a given tRNA specificity on the basis of phylogenetic relationships or on the basis of structural peculiarities. Mamit-tRNA further displays static individual 2D structures of human mitochondrial tRNA genes with location of polymorphisms and pathology-related point mutations. The site offers also a table allowing for an easy conversion of human mitochondrial genome nucleotide numbering into conventional tRNA numbering. The database is expected to facilitate exploration of structure/function relationships of mitochondrial tRNAs and to assist clinicians in the frame of pathology-related mutation assignments.     

Structural and functional accommodation of nucleotide variations at a conserved tRNA tertiary base pair.

The U8:A14 tertiary base pair of transfer RNAs (tRNAs) stabilizes the sharp turn from the acceptor stem to the dihydrouridine stem. This tertiary base pair is important for the overall L-shaped tRNA structure. Inspection of tRNA sequences shows that U8:A14 is highly conserved. However, variations of U8:A14 are found in natural sequences. This raises the question of whether all 16 permutations of U8:A14 can be accommodated by a single tRNA sequence framework and by the bacterial translational apparatus. Here we expressed the wild type and 15 variants of U8:A14 of an alanine tRNA amber suppressor in Escherichia coli and tested the ability of each to suppress an amber mutation. We showed that 12 of the 15 variants are functional suppressors (sup+) and 3 are nonfunctional (sup-). Of the 12 functional suppressors, the G8:G14 variant is the most efficient suppressor, whose suppression efficiency is indistinguishable from that of the wild type. Analysis of tRNA structure with chemical probes and the lead-cleavage reaction, however, showed a distinct difference between the G8:G14 variant and the wild type. Thus, two different structures of E. coli tRNAAla/CUA share an identical functional phenotype in protein synthesis. The remaining 11 sup+ variants with reduced suppression efficiencies are likely to have other structural variations. We suggest that the variations of these sup+ mutants are structurally and functionally accommodated by the bacterial translational apparatus. In contrast, the three sup- mutants harbor variations that alter the backbone structure in the corner of the L. These variations are likely to reduce the stability of the tRNA inside the cell or, among others, to interfere with the ability of the tRNA to functionally interact with elongation factor Tu and with the ribosome.     

Avoidance of antisense, antiterminator tRNA anticodons in vertebrate mitochondria

Protein synthesis (translation) stops at stop codons, codons not complemented by tRNA anticodons. tRNAs matching stops, antitermination (Ter) tRNAs, prevent translational termination, producing dysfunctional proteins. Genomes avoid tRNAs with anticodons whose complement (the anticodon of the ‘antisense’ tRNA) matches stops. This suggests that antisense tRNAs, which also form cloverleaves, are occasionally expressed. Mitochondrial antisense tRNA expression is plausible, because both DNA strands are transcribed as single RNAs, and tRNA structures signal RNA maturation. Results describe potential antisense Ter tRNAs in mammalian mitochondrial genomes detected by tRNAscan-SE, and evidence for adaptations preventing translational antitermination: genomes possessing Ter tRNAs use less corresponding stop codons; antisense Ter tRNAs form weaker cloverleaves than homologuous non-Ter antisense tRNAs; and genomic stop codon usages decrease with stabilities of codon-anticodon interactions and of Ter tRNA cloverleaves. This suggests that antisense tRNAs frequently function in translation. Results suggest that opposite strand coding is exceptional in modern genes, yet might be frequent for mitochondrial tRNAs. This adds antisense tRNA templating to other mitochondrial tRNA functions: sense tRNA templating, formation and regulation of secondary (light strand DNA) replication origins. Antitermination probably affects mitochondrial degenerative diseases and ageing: pathogenic mutations are twice as frequent in tRNAs with antisense Ter anticodons than in other tRNAs, and species lacking mitochondrial antisense Ter tRNAs have longer mean maximal lifespans than those possessing antisense Ter tRNAs.     

Evolution of the mitochondrial protein synthetic machinery

Comparative analysis of the components of the mitochondrial translational apparatus reveals a remarkable variability. For example the mitochondrial ribosomal rRNAs, display a three-fold difference in size in different organisms as a result of insertions or deletions, which affect specific areas of the rRNA molecule. This suggests that such areas are either not essential for mitoribosome function or that they can be replaced by proteins. Also mitochondrial tRNAs and mitoribosomal proteins are much less conserved than their cytoplasmic counterparts. Not only do the mitochondrial translational molecules vary in properties, also the location of the genes from which they are derived is not the same in all cases: mitochondrial tRNA genes which usually are found in the mtDNA, may have a nuclear location in protozoa and, conversely, only in fungi one finds a mitoribosomal protein gene in the organellar genome. The high rate of change of the components of the mitochondrial protein synthesizing machinery is accompanied by a number of unique features of the translation process: (i) the mitochondrial genetic code differs substantially from the standard code in a species-specific manner; (ii) special codon-anticodon recognition rules are followed; (iii) unusual mechanisms of translational initiation may exist. These observations suggest that the evolutionary pressures that have shaped the present day mitochondrial translational apparatus have been different in different organisms and also distinct from those acting on the cytoplasmic machinery. In spite of the interspecies variability, however, many features of the mitochondrial and bacterial protein synthetic apparatus show a clear resemblance, providing support for the hypothesis of a prokaryotic endosymbiont ancestry of mitochondria.     

Identification and characterization of mammalian mitochondrial tRNA nucleotidyltransferases

The CCA-adding enzyme (ATP:tRNA adenylyltransferase or CTP:tRNA cytidylyltransferase (EC )) generates the conserved CCA sequence responsible for the attachment of amino acid at the 3' terminus of tRNA molecules. It was shown that enzymes from various organisms strictly recognize the elbow region of tRNA formed by the conserved D- and T-loops. However, most of the mammalian mitochondrial (mt) tRNAs lack consensus sequences in both D- and T-loops. To characterize the mammalian mt CCA-adding enzymes, we have partially purified the enzyme from bovine liver mitochondria and determined cDNA sequences from human and mouse dbESTs by mass spectrometric analysis. The identified sequences contained typical amino-terminal peptides for mitochondrial protein import and had characteristics of the class II nucleotidyltransferase superfamily that includes eukaryotic and eubacterial CCA-adding enzymes. The human recombinant enzyme was overexpressed in Escherichia coli, and its CCA-adding activity was characterized using several mt tRNAs as substrates. The results clearly show that the human mt CCA-adding enzyme can efficiently repair mt tRNAs that are poor substrates for the E. coli enzyme although both enzymes work equally well on cytoplasmic tRNAs. This suggests that the mammalian mt enzymes have evolved so as to recognize mt tRNAs with unusual structures.     

Mitochondrial tRNAs in the lower fungus Spizellomyces punctatus: tRNA editing and UAG 'stop' codons recognized as leucine.

The mitochondrial DNA of the chytridiomycete fungus Spizellomyces punctatusen codes only eight tRNAs, although a minimal set of 24-25 tRNAs is normally found in fungi. One of these tRNAs has a CAU anticodon and is structurally related to leucine tRNAs, which would permit the translation of the UAG 'stop' codons that occur in most of its protein genes. The predicted structures of all S. punctatus tRNAs have the common feature of containing one to three mis-pairings in the first three positions of their acceptor stems. Such mis-pairing is expected to impair proper folding and processing of tRNAs from their precursors. Five of these eight RNAs were shown to be edited at the RNA level, in the 5'portion of the molecules. These changes include both pyrimidine to purine and A to G substitutions that restore normal pairing in the acceptor stem. Editing was not found at other positions of the tRNAs, or in the mitochondrial mRNAs of S. punctatus. While tRNA editing has not been observed in other fungi, the editing pattern inS.punctatus is virtually identical to that described in the amoeboid protozoan Acanthamoeba castellanii. If this type of mitochondrial tRNA editing has originated from their common ancestor, one has to assume that it was independently lost in plants, animals and in most fungi. Alternatively, editing might have evolved independently, or the genes coding for the components of the editing machinery were laterally transferred.     

Evolution of Pulmonate Gastropod Mitochondrial Genomes: Comparisons of Gene Organizations of Euhadra, Cepaea and Albinaria and Implications of Unusual Trna Secondary Structures

Complete gene organizations of the mitochondrial genomes of three pulmonate gastropods, Euhadra herklotsi, Cepaea nemoralis and Albinaria coerulea, permit comparisons of their gene organizations. Euhadra and Cepaea are classified in the same superfamily, Helicoidea, yet they show several differences in the order of tRNA and protein coding genes. Albinaria is distantly related to the other two genera but shares the same gene order in one part of its mitochondrial genome with Euhadra and in another part with Cepaea. Despite their small size (14.1-14.5 kbp), these snail mtDNAs encode 13 protein genes, two rRNA genes and at least 22 tRNA genes. These genomes exhibit several unusual or unique features compared to other published metazoan mitochondrial genomes, including those of other molluscs. Several tRNAs predicted from the DNA sequences possess bizarre structures lacking either the T stem or the D stem, similar to the situation seen in nematode mt-tRNAs. The acceptor stems of many tRNAs show a considerable number of mismatched basepairs, indicating that the RNA editing process recently demonstrated in Euhadra is widespread in the pulmonate gastropods. Strong selection acting on mitochondrial genomes of these animals would have resulted in frequent occurrence of the mismatched basepairs in regions of overlapping genes.     

Search for characteristic structural features of mammalian mitochondrial tRNAs

A number of mitochondrial (mt) tRNAs have strong structural deviations from the classical tRNA cloverleaf secondary structure and from the conventional L-shaped tertiary structure. As a consequence, there is a general trend to consider all mitochondrial tRNAs as "bizarre" tRNAs. Here, a large sequence comparison of the 22 tRNA genes within 31 fully sequenced mammalian mt genomes has been performed to define the structural characteristics of this specific group of tRNAs. Vertical alignments define the degree of conservation/variability of primary sequences and secondary structures and search for potential tertiary interactions within each of the 22 families. Further horizontal alignments ascertain that, with the exception of serine-specific tRNAs, mammalian mt tRNAs do fold into cloverleaf structures with mostly classical features. However, deviations exist and concern large variations in size of the D- and T-loops. The predominant absence of the conserved nucleotides G18G19 and T54T55C56, respectively in these loops, suggests that classical tertiary interactions between both domains do not take place. Classification of the tRNA sequences according to their genomic origin (G-rich or G-poor DNA strand) highlight specific features such as richness/poorness in mismatches or G-T pairs in stems and extremely low G-content or C-content in the D- and T-loops. The resulting 22 "typical" mammalian mitochondrial sequences built up a phylogenetic basis for experimental structural and functional investigations. Moreover, they are expected to help in the evaluation of the possible impacts of those point mutations detected in human mitochondrial tRNA genes and correlated with pathologies.     

A bacterial amber suppressor in Saccharomyces cerevisiae is selectively recognized by a bacterial aminoacyl-tRNA synthetase.

Little is known about the conservation of determinants for the identities of tRNAs between organisms. We showed previously that Escherichia coli tyrosine tRNA synthetase can charge the Saccharomyces cerevisiae mitochondrial tyrosine tRNA in vivo, even though there are substantial sequence differences between the yeast mitochondrial and bacterial tRNAs. The S. cerevisiae cytoplasmic tyrosine tRNA differs in sequence from both its yeast mitochondrial and E. coli counterparts. To test whether the yeast cytoplasmic tyrosyl-tRNA synthetase recognizes the E. coli tRNA, we expressed various amounts of an E. coli tyrosine tRNA amber suppressor in S. cerevisiae. The bacterial tRNA did not suppress any of three yeast amber alleles, suggesting that the yeast enzymes retain high specificity in vivo for their homologous tRNAs. Moreover, the nucleotides in the sequence of the E. coli suppressor that are not shared with the yeast cytoplasmic tyrosine tRNA do not create determinants which are efficiently recognized by other yeast charging enzymes. Therefore, at least some of the determinants that influence in vivo recognition of the tyrosine tRNA are specific to the cell compartment and organism. In contrast, expression of the cognate bacterial tyrosyl-tRNA synthetase together with the bacterial suppressor tRNA led to suppression of all three amber alleles. The bacterial enzyme recognized its substrate in vivo, even when the amount of bacterial tRNA was less than about 0.05% of that of the total cytoplasmic tRNA.     

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases.

The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined. Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element. However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E. coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase. Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E. coli. tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem. In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme. Moreover, enlarging the size of the anticodon loop of E. coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase. Finally, E. coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C. This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met).     

Expression of bovine mitochondrial tRNASer GCU derivatives in Escherichia coli.

By replacing a stretch of five A-U base pairs in the acceptor stem with G-C pairs, mitochondrial tRNA-SerGCU lacking a D arm could be expressed in Escherichia coli cells in considerable amounts. The expressed tRNA with no modified nucleoside was serylated in vitro with the mitochondrial enzyme. The tRNASerGCU derivatives carrying identity elements for alanine tRNA and the related anticodons were expressed. However, this expression event did not affect cell growth, probably because the expression started from the late log phase, which suggests that these mitochondrial tRNA derivatives are not involved in E.coli gene expression systems. Although there are some restrictions in the secondary structure of tRNAs that can be expressed by this method, it could prove useful for preparing large amounts of heterologous tRNAs in vivo.     

Pathogenic mutations in antisense mitochondrial tRNAs

Pathogenic mutations in mitochondrial tRNAs are 6.5 times more frequent than in other mitochondrial genes. This suggests that tRNA mutations perturb more than one function. A potential additional tRNA gene function is that of templating for antisense tRNAs. Pathogenic mutations weaken cloverleaf secondary structures of sense tRNAs. Analyses here show similar effects for most antisense tRNAs, especially after adjusting for associations between sense and antisense cloverleaf stabilities. These results imply translational activity by antisense tRNAs. For sense tRNAs Ala and Ser UCN, pathogenicity associates as much with sense as with antisense cloverleaf formation. For tRNA Pro, pathogenicity seems associated only with antisense, not sense tRNA cloverleaf formation. Translational activity by antisense tRNAs is expected for the 11 antisense tRNAs processed by regular sense RNA maturation, those recognized by their cognate amino acid’s tRNA synthetase, and those forming relatively stable cloverleaves as compared to their sense counterpart. Most antisense tRNAs probably function routinely in translation and extend the tRNA pool (extension hypothesis); others do not (avoidance hypothesis). The greater the expected translational activity of an antisense tRNA, the more pathogenic mutations weaken its cloverleaf secondary structure. Some evidence for RNA interference, a more classical role for antisense tRNAs, exists only for tRNA Ser UCN. Mutation pathogenicity probably frequently results from a mixture of effects due to sense and antisense tRNA translational activity for many mitochondrial tRNAs. Genomic studies should routinely explore for translational activity by antisense tRNAs.     

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.     

28 The study of tRNA modifications by molecular dynamics

Modified nucleosides are prevalent in tRNA. Experimental studies reveal that modifications play an important role in tuning tRNA activity. In this study, molecular dynamics (MD) simulations were used to investigate how modifications alter tRNA structure and dynamics. The X-ray crystal structures of tRNA-Asp, tRNA-Phe, and tRNA-iMet, both with and without modifications, were used as initial structures for 333-ns time-scale MD trajectories with AMBER. For each tRNA molecule, three independent trajectory calculations were performed. Force field parameters were built using the RESP procedure of Cieplak et al. for 17 nonstandard tRNA residues. The global root-mean-square deviations (RMSDs) of atomic positions show that modifications only introduce significant rigidity to tRNA-Phe’s global structure. Interestingly, regional RMSDs of anticodon stem-loop suggest that modified tRNA has more rigid structure compared to the unmodified tRNA in this domain. The anticodon RMSDs of the modified tRNAs, however, are higher than those of corresponding unmodified tRNAs. These findings suggest that rigidity of the anticodon arm is essential for tRNA translocation in the ribosome complex, and, on the other hand, flexibility of anticodon might be critical for anticodon–codon recognition. We also measure the angle between the 3D L-shaped arms of tRNA; backbone atoms of acceptor stem and TψC stem loop are selected to indicate one vector, and backbone atoms of anticodon stem and D stem loop are selected to indicate the other vector. By measuring the angle between two vectors, we find that the initiator tRNA has a narrower range of hinge motion compared to tRNA-Asp and tRNA-Phe, which are elongator tRNA. This suggests that elongator tRNAs, which might require significant flexibility in this hinge to transition from the A–to-P site in the ribosome, have evolved to specifically accommodate this need.     

Relation between tRNase activity and the structure of colicin D according to X-ray crystallography

Colicin D is a plasmid-encoded proteinaceous toxin which kills sensitive Escherichia coli. Toxicity stems from ribonuclease activity that targets exclusively four isoacceptors of tRNA(Arg) with a cleavage position between 38 and 39 of the corresponding anticodons. Since no other tRNAs with the same sequences at 38 and 39 as tRNA(Arg)s are cleaved, colicin D should be capable of recognizing some higher order structure of tRNAs. We report here two crystal structures of catalytic domains of colicin D which have different N-terminal lengths, both complexed with its cognate inhibitor protein, ImmD. A row of positive charge patches is found on the surface of the catalytic domain, suggestive of the binding site of the tRNAs. This finding, together with our refined tRNase activity experiments, indicates that the catalytic domain starting at position 595 has activity almost equivalent to that of colicin D.     

Pocketknife tRNA hypothesis: Anticodons in mammal mitochondrial tRNA side-arm loops translate proteins?

Peptide elongation proceeds by tRNA anticodons recognizing mRNA codons coding for the tRNA's cognate amino acid. Putatively, tRNAs possess three anticodons because tRNA side and anticodon-arms form similar stem-loop structures. Two lines of evidence indicate that mammal mitochondrial tRNA sidearms function as anticodons: numbers of TΨC-arm ‘anticodons’ matching specific cognates coevolve with that cognate's usage in mitochondrial genomes; and predicted ‘tetragene’ numbers, genes coded by quadruplet codons (tetracodons), coevolve with numbers of expanded anticodons in D-arms, as previously observed between tetragenes and antisense tRNA expanded anticodons. Sidearms with long stems and high GC contents contribute most to tRNA sidearm-tetragene coevolution. Results are compatible with two hypothetical mechanisms for translation by side-arms: crossovers exchange anticodon- and side-arms; tRNA sidearms are excised, aminoacylated and function as isolated stem-loop hairpins (more probable for long, respectively stable branches). Isolated sidearms would resemble recently described armless ‘minimal’ tRNAs. Isolated hairpins might most parsimoniously explain observed patterns. tRNA genes templating for three, rather than one functional tRNA, compress minimal genome size. Results suggest fused tRNA halves form(ed) modern tRNAs, isolated tRNA subparts occasionally translate proteins. Results confirm translational activity by antisense tRNAs, whose anticodons also coevolve with codon usages. Accounting for antisense anticodons improves results for sidearm anticodons.     

Evolutionary coadaptation of the motif 2--acceptor stem interaction in the class II prolyl-tRNA synthetase system

Known crystal structures of class II aminoacyl-tRNA synthetases complexed to their cognate tRNAs reveal that critical acceptor stem contacts are made by the variable loop connecting the beta-strands of motif 2 located within the catalytic core of class II synthetases. To identify potential acceptor stem contacts made by Escherichia coli prolyl-tRNA synthetase (ProRS), an enzyme of unknown structure, we performed cysteine-scanning mutagenesis in the motif 2 loop. We identified an arginine residue (R144) that was essential for tRNA aminoacylation but played no role in amino acid activation. Cross-linking experiments confirmed that the end of the tRNA(Pro) acceptor stem is proximal to this motif 2 loop residue. Previous work had shown that the tRNA(Pro) acceptor stem elements A73 and G72 (both strictly conserved among bacteria) are important recognition elements for E. coli ProRS. We carried out atomic group "mutagenesis" studies at these two positions of E. coli tRNA(Pro) and determined that major groove functional groups at A73 and G72 are critical for recognition by ProRS. Human tRNA(Pro), which lacks these elements, is not aminoacylated by the bacterial enzyme. An analysis of chimeric tRNA(Pro) constructs showed that, in addition to A73 and G72, transplantation of the E. coli tRNA(Pro) D-domain was necessary and sufficient to convert the human tRNA into a substrate for the bacterial synthetase. In contrast to the bacterial system, base-specific acceptor stem recognition does not appear to be used by human ProRS. Alanine-scanning mutagenesis revealed that motif 2 loop residues are not critical for tRNA aminoacylation activity of the human enzyme. Taken together, our results illustrate how synthetases and tRNAs have coadapted to changes in protein-acceptor stem recognition through evolution.     

Structure and function of initiator methionine tRNA from the mitochondria of Neurospora crassa.

Initiator methionine tRNA from the mitochondria of Neurospora crassa has been purified and sequenced. This mitochondrial tRNA can be aminoacylated and formylated by E. coli enzymes, and is capable of initiating protein synthesis in E. coli extracts. The nucleotide composition of the mitochondrial initiator tRNA (the first mitochondrial tRNA subjected to sequence analysis) is very rich in A + U, like that reported for total mitochondrial tRNA. In two of the unique features which differentiate procaryotic from eucaryotic cytoplasmic initiator tRNAs, the mitochondrial tRNA appears to resemble the eucaryotic initiator tRNAs. Thus unlike procaryotic initiator tRNAs in which the 5′ terminal nucleotide cannot form a Watson-Crick base pair to the fifth nucleotide from the 3′ end, the mitochondrial tRNA can form such a base pair; and like the eucaryotic cytoplasmic initiator tRNAs, the mitochondrial initiator tRNA lacks the sequence -TΨCG(or A) in loop IV. The corresponding sequence in the mitochondrial tRNA, however, is -UGCA- and not -AU(or Ψ)CG-as found in all eucaryotic cytoplasmic initiator tRNAs. In spite of some similarity of the mitochondrial initiator tRNA to both eucaryotic and procaryotic initiator tRNAs, the mitochondrial initiator tRNA is basically different from both these tRNAs. Between these two classes of initiator tRNAs, however, it is more homologous in sequence to procaryotic (56–60%) than to eucaryotic cytoplasmic initiator tRNAs (45–51%).