Normal and Mutant Glycine Transfer RNAs

THE glycine-specific tRNAs of E. coli can be grouped into three subspecies which are separated by chromatography on benzoylated DEAE cellulose (BDC): tRNA^Gly₁ (GGG), tRNA^Gly₂ (GGA/G) and tRNA^Gly₃ (GGU/C)^1,2. The tRNA^Gly₁ and tRNA^Gly₂ are specified by the genes, glyU and glyT, respectively, which have been located at 55 and 77 minutes on the E. coli chromosome. Suppressors of tryptophan A gene (trpA) missense mutations and partial diploid strains have been used extensively to characterize the glycine tRNA structural genes (Table 1)^1–3. A common property of these suppressor mutations is that the altered tRNA^Gly is no longer aminoacylated at the normal rate by the glycyl tRNA synthetase (GRS). When ordinary loading conditions are used virtually none of the suppressor tRNA species are amino-acylated. These studies have shown that single gene copies are normally present at the glyT and glyU loci.     

Activation of several tRNAs of Escherichia coli by the phenoxyacetyl derivative of N-hydroxysuccinimide

Escherichia coli 15T^? treated with chloramphenicol produces tRNA^phe which is deficient in minor nucleosides. Undermodified tRNA^phe chromatographs as two new peaks from a benzoylated diethylaminoethyl-cellulose column. Chloramphenicol tRNA^phe was purified by phenoxyacetylation of phenylalanyl-tRNA and subsequent chromatography on benzoylated diethylaminoethyl-cellulose. Purified tRNA^phe had an altered Chromatographie profile as a result of the purification procedure. Phenoxyacetylation of an unpurified tRNA preparation, which was either charged with phenylalanine or kept discharged, resulted in a permanent alteration of tRNA^phe which was similar to the alteration of the purified tRNA^phe. The altered tRNAs eluted with higher salt or ethanol concentrations from benzoylated diethylaminoethyl-cellulose. The alteration was also shown for tRNA^phe of phenoxyacetylated tRNA from late log phase E. coli 15T?. tRNA^glu and tRNA^Leu were not changed, but both tRNA^Arg and tRNA^Ile were altered. tRNA₂^Val and tRNA^Met shifted in the elution profile; tRNA₁^Val and tRNA_f^Met were not affected.Comparison of the primary structures of the alterable and nonalterable tRNA's revealed that all alterable tRNA's have the undefined nucleoside X in the extra loop. Phenoxyacetylation of nucleoside X probably was the cause of the altered profiles.tRNA^phe from E. coli 15T^? treated with chloramphenicol was less reactive towards phenoxyacetylation than normal tRNA, possibly because of a different conformation of the modification-deficient molecule relative to the normal tRNA^phe. tRNA^phe from E. coli 15T^?, starved for cysteine and methionine and treated with chloram-phenicol, is more deficient in minor nucleosides and showed even less reactivity.Acceptor capacities of the altered tRNA species were not changed significantly; only the acceptor capacity for tRNA^Ile decreased approximately 25%. The recognition site for the aminoacyl-tRNA synthetases probably is not affected.     

Procedure for purification of recombinant preMsk1p from <Emphasis Type="Italic">E. coli</Emphasis> determines its properties as a factor of tRNA import into yeast mitochondria

Mitochondrial genomes of many eukaryotic organisms do not code for the full tRNA set necessary for organellar translation. Missing tRNA species are imported from the cytosol. In particular, one out of two cytosolic lysine tRNAs of the yeast Saccharomyces cerevisiae is partially internalized by mitochondria. The key protein factor of this process is the precursor of mitochondrial lysyl-tRNA synthetase, preMsk1p. In this work, we show that recombinant preMsk1p purified from E. coli in native conditions, when used in an in vitro tRNA import system, demonstrates some properties different from those shown by the renatured protein purified from E. coli in the denatured state. We also discuss the possible mechanistic reasons for this phenomenon.     

Mutant tRNA<Superscript>His</Superscript> Ineffective in Repression and Lacking Two Pseudouridine Modifications

TRANSFER RNA has been implicated in the regulation of a number of amino-acid biosynthetic operons^1–4. Histidyl-tRNA^His has been shown to be involved in regulation of the histidine operon by analysis of six genes (hisO, hisR, hisS, hisT, hisU, hisW), mutation of which causes derepression of the enzymes of the histidine biosynthetic pathway in Salmonella typhimurium^5–7. A class of derepressed mutants (hisR) has only about 55% as much tRNA^His as the wild type⁴ and in the one example sequenced, contains tRNA^HIS with a structure identical to that of the wild type⁸. Studies of mutants of the gene for histidyl-tRNA synthetase (hisS) indicated that the derepressed phenotype was associated with defects in the charging of tRNA^HISin vitro². The amounts of charged and uncharged tRNA^His present in vivo during physiological derepression of the wild type and in the six classes of regulatory mutants, have been determined⁹. This work has shown that repression of the histidine operon is correlated directly with the concentration of charged histidyl-tRNA^Hisin vivo and not with the ratio of charged to uncharged or the absolute amount of uncharged tRNA^His. The derepression observed in mutants, of hisS (the gene for histidyl-tRNA synthetase), hisR (the presumed structural gene for the single species of tRNA^His) and hisU and hisW (genes presumably involved in tRNA modification) may be explained by the lower cellular concentration of charged tRNA^His which these mutants contain.     

Sequence Relationship of Three Valine Acceptor tRNAs from <Emphasis Type="Italic">Escherichia coli</Emphasis>

THE degree of degeneracy of the genetic code varies for the twenty amino-acids: between one and six different triplets are assigned to a single amino-acid. Four triplets GUU, GUC, GUA, GUG code for the amino-acid valine^1,2. Two valine specific tRNAs have been separated by fractionation of mixed E. coli tRNA³; tRNA^val₁ is specific for GU^A_G and tRNA^val₂ corresponds to GU^U_C (see also ref. 1 for binding properties). Recent studies showed that although both species are recognized by the single activating enzyme present in E. coli, the association constant (Ka) for the minor species, tRNA^val₂ (?20% of total acceptor), is an order of magnitude higher than the association constant of the major species, tRNA^{val 4}₁. As a first step to comparing the structures of these two tRNAs, we analysed the base sequences of the major and minor species. We recently published the nucleotide sequence of tRNA^{val 5}₁; we report here the sequence of two minor subspecies (quite similar to each other) that comprise the tRNA^val₂ acceptor and we comment on the significance of the sequence homologies in relation to the problems of enzyme recognition and tRNA evolution.     

Functional replacement of the endogenous tyrosyl-tRNA synthetase–tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion

Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase–tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)–tRNA^Tyr pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS–tRNA^Tyr pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNA^Tyr. The endogenous TyrRS and tRNA^Tyr genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS–tRNA^Tyr pair. In this engineered strain, 3-iodo-l-tyrosine and 3-azido-l-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-l-tyrosine and was also found to recognize 3-azido-l-tyrosine. The structural basis for the 3-azido-l-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.     

Suppression of Amber Codons in Caulobacter crescentus by the Orthogonal Escherichia coli Histidyl-tRNA Synthetase/tRNAHis Pair

While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium Caulobacter crescentus. Based on a previous report that C. crescentus and Escherichia coli tRNA^His have distinctive identity elements, we constructed E. coli tRNA^His _CUA, a UAG suppressor tRNA for C. crescentus. By examining the expression of three UAG codon- containing reporter genes (encoding a β-lactamase, the fluorescent mCherry protein, or the C. crescentus xylonate dehydratase), we demonstrated that the E. coli histidyl-tRNA synthetase/tRNA^His _CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNA^His _CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNA^His _CUA pair is orthogonal in C. crescentus. These results illustrate that UAG suppression can be achieved in C. crescentus with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair.     

Aminoacylation of Bisulphite-modified Yeast Tyrosine Transfer RNA

AS part of a search for the chemical rules underlying the recognition of a tRNA by its aminoacyl-tRNA ligase¹, we are examining the effect of C?U transitions on aminoacylation of tRNA. In this paper, we describe four well-characterized modifications of yeast tRNA^Tyr induced by reaction with bisulphite ions at pH 5.8. Three of the observed changes proved to be C-?U transitions produced by the series of reactions shown in Fig. 1 (refs. 2–5).     

The crystal structure of a Thermus thermophilus tRNA acceptor stem microhelix at 1.6 Å resolution

tRNAs are aminoacylated with the correct amino acid by the cognate aminoacyl-tRNA synthetase. The tRNA/synthetase systems can be divided into two classes: class I and class II. Within class I, the tRNA identity elements that enable the specificity consist of complex sequence and structure motifs, whereas in class II the identity elements are assured by few and simple determinants, which are mostly located in the tRNA acceptor stem.The tRNA^Gly/glycyl-tRNA-synthetase (GlyRS) system is a special case regarding evolutionary aspects. There exist two different types of GlyRS, namely an archaebacterial/human type and an eubacterial type, reflecting the evolutionary divergence within this system. We previously reported the crystal structures of an Escherichia coli and of a human tRNA^Gly acceptor stem microhelix. Here we present the crystal structure of a thermophilic tRNA^Gly aminoacyl stem from Thermus thermophilus at 1.6 Å resolution and provide insight into the RNA geometry and hydration.     

Overproduction and purification ofEscherichia coli tRNALeu

Chemically synthesized genes encodingEscherichia coli tRNA ₁ ^Leu and tRNA ₂ ^Leu were ligated into the plasmid pTrc99B. then transformed intoEscherichia coli MT102, respectively. The positive transformants, named MT-Leu1 and MT-Leu2, were confirmed by DNA sequencing, and the conditions of cultivation for the two transformants were optimized. As a result, leucinc accepting activity of their total tRNA reached 810 and 560 pmol/A₂₆₀, respectively: the content of tRNA ₁ ^Leu was 50% of total tRNA from MT-Leu1, while that of tRNA ₂ ^Leu was 30% of total tRNA from MT-Leu2. Both tRNA^Leus from their rotal tRNs were fractionated to 1 600 pmol/A₂₆₀ after DEAE-Sepharose and BD-cellulose column chromatography. The accurate kinetic constants of aminoacylation of the two isoacceptors of tRNA^Leu catalyzed by leucyl-tRNA synthetase were determined.     

Dual Targeting of a tRNAAsp Requires Two Different Aspartyl-tRNA Synthetases in Trypanosoma brucei

The mitochondrion of the parasitic protozoon Trypanosoma brucei does not encode any tRNAs. This deficiency is compensated for by partial import of nearly all of its cytosolic tRNAs. Most trypanosomal aminoacyl-tRNA synthetases are encoded by single copy genes, suggesting the use of the same enzyme in the cytosol and in the mitochondrion. However, the T. brucei genome encodes two distinct genes for eukaryotic aspartyl-tRNA synthetase (AspRS), although the cell has a single tRNA^Asp isoacceptor only. Phylogenetic analysis showed that the two T. brucei AspRSs evolved from a duplication early in kinetoplastid evolution and also revealed that eight other major duplications of AspRS occurred in the eukaryotic domain. RNA interference analysis established that both Tb-AspRS1 and Tb-AspRS2 are essential for growth and required for cytosolic and mitochondrial Asp-tRNA^Asp formation, respectively. In vitro charging assays demonstrated that the mitochondrial Tb-AspRS2 aminoacylates both cytosolic and mitochondrial tRNA^Asp, whereas the cytosolic Tb-AspRS1 selectively recognizes cytosolic but not mitochondrial tRNA^Asp. This indicates that cytosolic and mitochondrial tRNA^Asp, although derived from the same nuclear gene, are physically different, most likely due to a mitochondria-specific nucleotide modification. Mitochondrial Tb-AspRS2 defines a novel group of eukaryotic AspRSs with an expanded substrate specificity that are restricted to trypanosomatids and therefore may be exploited as a novel drug target.In most animal and fungal mitochondria, the total set of tRNAs required for translation is encoded on the mitochondrial genome and thus of bacterial evolutionary origin. The aminoacyl-tRNA synthetases (aaRSs)² responsible for charging of mitochondrial tRNAs are always nuclear encoded and need to be imported into mitochondria. We therefore expect to find two sets of aaRSs, one for cytosolic aminoacyl-tRNA synthesis and a second one, of bacterial evolutionary origin, for aminoacylation of mitochondrial tRNAs (1, 2).In most cells, however, some aaRSs are targeted to both the cytosol as well as to mitochondria (3). In Saccharomyces cerevisiae, for example, four aaRSs are double-targeted to both compartments, indicating that they are able to aminoacylate tRNAs of both eukaryotic and bacterial evolutionary origin (4–6). In plants, the situation is more complex, since protein synthesis occurs in three compartments: the cytosol, the mitochondria, and the plastids. A recent analysis in Arabidopsis has shown that, rather than having three unique sets of aaRSs specific for the three translation systems, more than 15 aaRSs were dually targeted to the mitochondria and the plastid (7). Moreover, there is at least one aaRS that is shared between all three compartments. In summary, these examples indicate that the overlap between the different sets of aaRSs used in the various translation systems is variable and can be extensive.Most eukaryotes, except many animals and fungi, lack a variable number of mitochondrial tRNA genes. Mitochondrial translation in these organisms depends on import of a small fraction of the corresponding nucleus-encoded cytosolic tRNAs (8–10). As a consequence, imported tRNAs are always of eukaryotic evolutionary origin. An intriguing situation is found in trypanosomatids (such as Trypanosoma brucei and Leishmania spp.), where all mitochondrial tRNA genes have apparently been lost and all mitochondrial tRNAs are imported from the cytosol. In these organisms, all mitochondrial tRNAs derive from cytosolic tRNAs (11). It is therefore reasonable to assume that trypanosomal aaRSs are dually targeted to the cytosol and the mitochondrion. For the T. brucei glutaminyl-tRNA synthetase (GlnRS) and the glutamyl-tRNA synthetase, the dual localization has been shown experimentally (12). Moreover, dual targeting of essentially all aaRSs is suggested by the fact that the genome of T. brucei and other trypanosomatids encodes only 23 distinct aaRSs, fewer than any other eukaryote that has a mitochondrial translation system (13). Unexpectedly, two distinct genes were found for the tryptophanyl-tRNA synthetase (TrpRS), the lysyl-tRNA synthetase and the aspartyl-tRNA synthetase (AspRS). A recent study has shown that the two trypanosomal TrpRSs are required for cytosolic and mitochondrial tryptophanyl-tRNA formation (14). Trypanosomal tRNA^Trp is imported to the mitochondria, where it undergoes C to U editing at the wobble nucleotide and is thiolated at position 33. The RNA editing is required to decode the reassigned mitochondrial tryptophan codon UGA (14–16). Both nucleotide modifications are antideterminants for the cytosolic TrpRS (14). As we concluded previously (14), the presence of a second TrpRS with expanded substrate specificity is required to efficiently aminoacylate imported, mature tRNA^Trp in trypanosomal mitochondria.The present study focuses on the characterization and functional analysis of another pair of duplicated trypanosomal aaRSs, the AspRSs. We show that the two enzymes are individually essential for normal growth of insect stage T. brucei. We also demonstrate that the two trypanosomal AspRSs are of eukaryotic evolutionary origin and that the aminoacylation of the cytosolic and mitochondrial tRNA^Asp species requires these two distinct AspRSs.     

Dual specificity of su+7 tRNA. Evidence for translational discrimination

The su⁺7 amber suppressor of Escherichia coli is a mutant tRNA^Trp that translates UAG codons as glutamine. Nevertheless, the purified su⁺7 tRNA can be charged with either glutamine or tryptophan. Aminoacylation kinetics in vitro suggest that the tRNA should be acylated with equal amounts of glutamine and tryptophan in vivo. The predominance of the glutamine specificity of the suppressor is therefore potentially anomalous. We can find no selective deacylation of tryptophanyl-su⁺7 tRNA by glutaminyl-tRNA synthetase, tryptophanyl-tRNA synthetase, or any other cellular element. Furthermore, as predicted, nearly equal amounts of glutaminyl and tryptophanyl-su⁺7 tRNA are actually detected in aminoacyl-tRNA extracted from growing cells. We conclude that the translational apparatus somehow discriminates against tryptophanyl-su⁺7 tRNA at a step after synthesis of the two aminoacyl-tRNAs.     

Complete mitochondrial genome of <Emphasis Type="Italic">Ampittia dioscorides</Emphasis> (Lepidoptera: Hesperiidae) and its phylogenetic analysis

The complete mitochondrial genome of Ampittia dioscorides (Lepidoptera: Hesperiidae) was determined. The sequenced genome is a circular molecule of 15313 bp, containing 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and an A + T-rich region. The gene arrangements and transcribing directions are identical to those in most of the reported lepidopteran mitogenomes. The base composition of the whole genome and genes or regions are also similar to those in other lepidopteran species. All the PCGs are initiated by typical ATN codons; the exception being COI, which begins with a CGA codon. Eight genes (ND2, ATPase8, ATPase6, COIII, ND5, ND4L, ND6, and Cytb) end with a TAA stop codon, and two genes (ND1 and ND3) end with TAG. The remaining three genes (COI and COII, which end with TA-, and ND4, which ends with T-) have incomplete stop codons. All tRNAs have the typical clover-leaf structure of mitochondrial tRNAs, with the exception of tRNA^Ser(AGY). On the basis of the concatenated nucleotide and amino acid sequences of the 13 PCGs and wingless gene of 22 butterfly species, maximum parsimony (MP) and Bayesian inference (BI) trees were constructed, respectively. Both MP and BI trees had the same topological structure: ((((Nymphalidae + Danaidae) + Lycaenidae) + Pieridae) + Papilionidae) + Hesperiidae). The results provide support for Hesperiidae as a superfamily-level taxon.     

Interaction of yeast arginyl-tRNA synthetase and aspartyl-tRNA synthetase with Blue-dextran Sepharose : assignment of the Blue-Dextran Binding site on the synthetases

Yeast arginyl-tRNA synthetase and aspartyl-tRNA synthetase like nucleotidyl transferases previously investigated interact with the Blue-Dextran-Sepharose affinity ligand through their tRNA binding domain: the enzymes are readily displaced from the affinity column by their cognate tRNAs but not by ATP or a mixture of ATP and the cognate amino acid in contrast to other aminoacyl-tRNA synthetases. In the absence of Mg⁺⁺, the arginyl-tRNA synthetase can be dissociated from the column by tRNA^Asp and tRNA^Phe which have been shown to be able to form a complex with the synthetase, but in presence of Mg⁺⁺ the elution is only obtained by the specific tRNA.The procedure described here can thus be used: (i) to detect polynucleotide binding sites in a protein; (ii) to estimate the relative affinities of different tRNAs for a purified synthetase; (iii) to purify an aminoacyl-tRNA synthetase by selective elution with the cognate tRNA.     

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.     

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.     

The Role of Initiator tRNAi met in Fidelity of Initiation of Protein Synthesis

The proper arrangement of amino acids in a protein determines its proper function, which is vital for the cellular metabolism. This indicates that the process of peptide bond formation requires high fidelity. One of the most important processes for this fidelity is kinetic proofreading. As biochemical experiments suggest that kinetic proofreading plays a major role in ensuring the fidelity of protein synthesis, it is not certain whether or not a misacylated tRNA would be corrected by kinetic proofreading during the peptide bond formation. Using 2-layered ONIOM (QM/MM) computational calculations, we studied the behavior of misacylated tRNAs and compared the results with these for cognate aminoacyl-tRNAs during the process of peptide bond formation to investigate the effect of nonnative amino acids on tRNAs. The difference between the behavior of initiator tRNA_i ^met compared to the one for the elongator tRNAs indicates that only the initiator tRNA_i ^met specifies the amino acid side chain.     

Genetically encoded amino acids with <Emphasis Type="Italic">tert</Emphasis>-butyl and trimethylsilyl groups for site-selective studies of proteins by NMR spectroscopy

The amino acids 4-(tert-butyl)phenylalanine (Tbf) and 4-(trimethylsilyl)phenylalanine (TMSf), as well as a partially deuterated version of Tbf (dTbf), were chemically synthesized and site-specifically incorporated into different proteins, using an amber stop codon, suppressor tRNA and the broadband aminoacyl-tRNA synthetase originally evolved for the incorporation of p-cyano-phenylalanine. The ¹H-NMR signals of the tert-butyl and TMS groups were compared to the ¹H-NMR signal of tert-butyltyrosine (Tby) in protein systems with molecular weights ranging from 8 to 54 kDa. The ¹H-NMR resonance of the TMS group appeared near 0 ppm in a spectral region with few protein resonances, facilitating the observation of signal changes in response to ligand binding. In all proteins, the R ₂ relaxation rate of the tert-butyl group of Tbf was only little greater than that of Tby (less than two-fold). Deuteration of the phenyl ring of Tbf made only a relatively small difference. The effective T ₂ relaxation time of the TMS signal was longer than 140 ms even in the 54 kDa system.     

Separation of aminoacyl-transfer RNAs by polyacrylamide gel electrophoresis

A polyacrylamide gel electrophoresis system for separating $\text{E.}$$\text{coli}$ tRNAs and aminoacyl-tRNAs is described. The tRNA was separated into 6 discrete bands which contained varyin aamounts of tRNA and therefore varying numbers of tRNA species. In order to locate specific tRNAs, tRNA was charged with a ¹⁴C amino acid and the aminoacyl-tRNA was located by autoradiography. With several amino acids, 2 isoaccepting species were found. In total, 30 aminoacyl-tRNAs were located.