Formation of chromatographically unique species of transfer ribonucleic acid during amino acid starvation of relaxed-control Escherichia coli.

Examination of the transfer ribonucleic acid (tRNA) produced by starving, relaxed-control (rel minus) strains of Escherichia coli for required amino acids revealed the occurrence of a number of chromatographically unique subspecies. Leucine starvation results in the formation of new isoacceptor species of leucine-, histidine-, arginine-, valine-, and phenylalanine-specific tRNA and quantitative changes in the column profiles of serine, glycine, and isoleucine tRNA. Evidence that the unique tRNA species are synthesized de novo during amino acid starvation comes from the findings that the major unique leucine isoacceptor species is not formed in stringent control cells or in rel minus cells starved for uracil or treated with rifampin. Furthermore, heat treatment of the unique leucine tRNA does not alter its chromatographic behavior, indicating that the species is not an aggregate or nuclease-damaged form of a normal isoacceptor tRNA. The methyl acceptor activities of tRNA from leucine-starved and nonstarved rel+ or rel minus cells were found to be essentially the same. This result and the finding that the chromatographic behavior of the unique leucine-specific tRNA was not altered after treatment with tRNA methylase suggests that gross methyl deficiency is probably not the biochemical basis for the occurrence of the unique species.     

Tests of the ribosomal editing hypothesis: amino acid starvation differentially enhances the dissociation of peptidyl-tRNA from the ribosome.

Starving Escherichia coli for amino acids affected the dissociation of peptidyl-tRNAs from ribosomes. The frequency of dissociation of specific peptidyl-tRNA families responded differently to starvation for different amino acids rather than uniformly to the general condition of starvation.These results are interpreted in terms of the ribosomal editing hypothesis Menninger 1977. Starvation for some aminoacyl-tRNAs resulted in more opportunities for other aminoacyl-tRNAs to err, providing a greater amount of erroneous peptidyl-tRNA to be dissociated by the ribosomal editor. The details of the response of particular peptidyl-tRNA families to particular amino acid starvations show that a tRNA less able to decode correctly as an aminoacyl-tRNA is more likely to dissociate from the ribosome after peptide transfer. Many of the errors of translation thought previously to be rare may not have been detected in completed proteins because the ribosomal editor is most active against them.The results can also be interpreted as a specific regulatory response to amino acid starvation by a ribosome forced to pause during translation of non-essential proteins at codons whose aminoacyl-tRNAs are limiting, a model known as translational triage.     

Age-related qualitative and quantitative changes in tRNA population of rat skeletal muscle

Total tRNA was purified from skeletal muscle of young, adult and old female albino rats. Age-dependent variation of total tRNA was the same with respect to tRNA content and biological activity as measured by amino acid acceptor capacity. The tRNA content was more in young rats and showed a gradual decrease in the adult and old rats. The relative abundancy of eleven aminoacyl-tRNAs were checked at each age and during aging. Arginyl, glutamyl and tyrosyl-tRNAs do not show any quantitative or qualitative change with age.     

Methyl-Deficient Transfer Ribonucleic Acid in Saccharomyces cerevisiae

Methyl-deficient transfer ribonucleic acid (tRNA) is found in certain methionine auxotrophs of Saccharomyces cerevisiae during logarithmic growth (at one generation time before the late growth phase) and during residual growth in the absence of exogenous methionine. The former effect seems to be accounted for by the general increase in RNA synthesis that occurs at the time; there is no specific synthesis of tRNA in the absence of ribosomal RNA synthesis, nor is the methyl group deficiency limited to a single tRNA species. During methionine starvation, all species of tRNA are methyl-deficient, but this occurs only in strains with certain blocks in the methionine pathway. The kinetics of disappearance of the methyl group donor, S-adenosylmethionine, during starvation of D73 (which accumulates methyl-deficient tRNA), do not differ from other strains, but D73 loses the methylase inhibitor, S-adenosylhomocysteine, much more slowly.     

Selective charging of tRNA isoacceptors induced by amino-acid starvation

Aminoacylated (charged) transfer RNA isoacceptors read different messenger RNA codons for the same amino acid. The concentration of an isoacceptor and its charged fraction are principal determinants of the translation rate of its codons. A recent theoretical model predicts that amino-acid starvation results in 'selective charging' where the charging levels of some tRNA isoacceptors will be low and those of others will remain high. Here, we developed a microarray for the analysis of charged fractions of tRNAs and measured charging for all Escherichia coli tRNAs before and during leucine, threonine or arginine starvation. Before starvation, most tRNAs were fully charged. During starvation, the isoacceptors in the leucine, threonine or arginine families showed selective charging when cells were starved for their cognate amino acid, directly confirming the theoretical prediction. Codons read by isoacceptors that retain high charging can be used for efficient translation of genes that are essential during amino-acid starvation. Selective charging can explain anomalous patterns of codon usage in the genes for different families of proteins.     

Control of protein synthesis in mammalian cells by aminoacylation of transfer ribonucleic acid.

The dependence of protein synthesis on the intracellular content of aminoacylated tRNA has been studied in mouse ascites tumor cells deprived for various amino acids. A remarkable reduction in net protein synthesis has been found only after a drastic decrease in aminoacylation of tRNA. The quantitative correlation of protein synthesis with the degree of aminoacylation suggests that a moderate amino acid starvation primarily influences the rate of elongation at the codon concerned. These results are in contrast to the findings previously reported for HeLa cells. Some crucial steps during the determination of intracellular aminoacyl-tRNA have been investigated. The reliability of the method employed has been discussed on a theoretical basis.     

A comparison of transfer RNA isoaccepting species between collagenous and noncollagenous tissues in the embryonic chick

Transfer RNA was isolated from different organs of 17-day-old chick embryos and the acceptor activity for each of the 20 amino acids was determined. The most abundant acceptor activities found in tRNA from tendon cells were for glycine, arginine, proline and alanine. When compared to the average acceptor activity found in brain, liver and heart, the tendon tRNA showed an increase in acceptor activity of 33% in glycine, 40% in arginine and 83% in proline. Reversed phase chromatography of the tRNA charged with glycine demonstrated that the increase in glycyl-tRNA in tendon could be accounted for by an increase in one of four major isoaccepting species. Such an increase in a single species was also observed in tRNA isolated from calvaria. The codon response of this species was shown to differ from that of the other glycyl-tRNA species. No major differences in the relative proportions of isoaccepting species could be demonstrated for any other amino acid. These results suggest that a characteristic complement of tRNA species may be associated with collagen synthesis.     

Aminoacylation of rat liver transfer RNA with homologous and heterologous enzyme systems during aging

Total tRNA extracted from livers of young (7 +/- 1 weeks), adult (40 +/- 1 weeks) and old (80 +/- 1 weeks) rats showed quantitative variation with age, being maximal in adults. Young and old animals yielded almost the same level of tRNAs. Quantitative changes in tRNAs were also observed from the study of amino acid acceptor activity using homologous enzyme i.e., aminoacyl-tRNA synthetase preparations from rat liver of the same age group. Quantitative variation followed the trend of qualitative variation. When tRNA was amino-acylated with a heterologous enzyme system, i.e., synthetase preparation from rat liver of another age group, age-related variation in aminoacyl-tRNA did not follow a pattern similar to that in the case of the homologous enzyme system. Young and adult synthetase enzymes showed maximum affinity for their homologous tRNAs but synthetases from old rat liver did not show any specific affinity for "old" tRNAs. This shows that apart from tRNAs, enzyme activity also changes with age.     

Rare transfer ribonucleic acid essential for phage growth. Nucleotide sequence comparison of normal and mutant T4 isoleucine-accepting transfer ribonucleic acid.

One of the eight tRNA species coded by bacteriophage T4 is unique in that (1) it is found in a yield lower by three- to fourfold than that of any other tRNA and (2) while dispensable for growth in standard laboratory hosts, it is essential for phage propagation in a natural isolate of Escherichia coli (strain CT439). We report here the nucleotide sequence of this tRNA and of several mutationally altered forms. The molecule is 77 nucleotides in length and has the anticodon N-A-U. Depending on the pairing properties of the "wobble" nucleotide N, this sequence could correspond to one or more of the isoleucine-specific codons (formula: see text) or to the methionine-specific codon A-U-G. Since a T4-specific acceptor activity for isoleucine which is stimulated in ribosome binding by A-U-A but not A-U-U has been reported previously, we infer that we have sequenced a tRNA Ile species which preferentially recognizes A-U-A. Mutant HA1 is unable to grow in CT439; it produces no tRNA Ile. The primary mutational alteration is a transition four residues from the 5'terminus which converts a C.G to a U.G base pair. The consequences of this lesion can be partially reversed by second-site mutations nearby in the acceptor stem. Unexpectedly, the tRNA Ile synthesized in these revertants still retains two unusual structural features found in the wild-type molecule: the opposition of two Up residues in the amino acid acceptor stem and the opposition of an Ap and a Gp residue in the anticodon stem. Implications of these structual anomalies for a possibly unique physiological role of this minor tRNA species are discussed.     

Isoaccepting lysine transfer ribonucleic acid species of Pseudomonas aeruginosa.

Pseudomonas aeruginosa tRNA was treated with iodine, CNBr and N-ethylmaleimide, three thionucleotide-specific reagents. Reaction with iodine resulted in extensive loss of acceptor activity by lysine tRNA, glutamic acid tRNA, glutamine tRNA, serine tRNA and tyrosine tRNA. CNBr treatment resulted in high loss of acceptor ability by lysine tRNA, glutamic acid tRNA and glutamine tRNA. Only the acceptor ability of tyrosine tRNA was inhibited up to 66% by N-ethylmaleimide treatment, a reagent specific for 4-thiouridine. By the combined use of benzoylated DEAE-cellulose and DEAE-Sephadex columns, lysine tRNA of Ps. aeruginosa was resolved into two isoaccepting species, a major, tRNA Lys1 and a minor, tRNALys1. Co-chromatography of 14C-labelled tRNALys1 and 3H-labelled tRNALys2 on benzoylated DEAE-cellulose at pH 4.5 gave two distinct, non-superimposable profiles for the two activity peaks, suggesting that they were separate species. The acceptor activity of these two species was inhibited by about 95% by iodine and CNBr. Both the species showed equal response to codons AAA and AAG and also for poly(A) and poly(A1,G1) suggesting that the anticodon of these species was UUU. Chemical modification of these two species by iodine did not inhibit the coding response. The two species of lysine of Ps. aeruginosa are truly redundant in that they are indistinguishable either by chemical modification or by their coding response.     

Characterization of the 42-S nucleoprotein particles of Cyprinus carpio oocytes

Previtellogenic oocytes of the fish Cyprinus carpio contain 42S nucleoprotein particles that are composed of two proteins of molecular weights 48,500 and 39,300 (molar ratio 2:1), tRNA and 5S RNA (molar ratio 3:1). The tRNA population embodied in the 42S particle contains all amino acid acceptor species but their distribution differs from that found in tRNA from mature oocytes.     

Initiation of in vivo protein synthesis with non-methionine amino acids

Methionine is the universal amino acid for initiation of protein synthesis in all known organisms. The amino acid is coupled to a specific initiator methionine tRNA by methionyl-tRNA synthetase. In Escherichia coli, attachment of methionine to the initiator tRNA (tRNA(fMet)) has been shown to be dependent on synthetase recognition of the methionine anticodon CAU (complementary to the initiation codon AUG), [Schulman, L. H., & Pelka, H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6755-6759]. We show here that alteration of the anticodon of tRNA(fMet) to GAC or GAA leads to aminoacylation of the initiator tRNA with valine or phenylalanine. In addition, tRNA(fMet) carrying these amino acids initiates in vivo protein synthesis when provided with initiation codons complementary to the modified anticodons. These results indicate that the sequence of the anticodon of tRNA(fMet) dictates the identity of the amino acid attached to the initiator tRNA in vivo and that there are no subsequent steps which prevent initiation of E. coli protein synthesis by valine and phenylalanine. The methods described here also provide a convenient in vivo assay for further examination of the role of the anticodon in tRNA amino acid acceptor identity.     

Purpuromycin: an antibiotic inhibiting tRNA aminoacylation.

Purpuromycin, an antibiotic produced by Actinoplanes ianthinogenes, had been reported previously to inhibit protein synthesis. In the present report, we demonstrate that the mechanism of action of this antibiotic is quite novel in that it binds with fairly high affinity to all tRNAs, inhibiting their acceptor capacity. Although more than one molecule of purpuromycin is bound to each tRNA molecule, the inhibitory activity of this antibiotic was found to be selective for the tRNA acceptor function; in fact, after the aminoacylation step, purpuromycin was found to affect none of the other tested functions of tRNA (interaction with the ribosomal P- and A-sites and interaction with translation factors). Accordingly, purpuromycin was found to inhibit protein synthesis only when translation depended on the aminoacylation of tRNA and not when the system was supplemented with pre-formed aminoacyl-tRNAs. Because purpuromycin did not interfere with the ATP-PPi exchange reaction of the synthetase or with the initial interaction of the enzyme with its tRNA substrate, the basis for the inhibition of aminoacylation is presumably the formation of a nonproductive synthetase-tRNA complex in the presence of purpuromycin in which the tRNA is unable to be charged with the corresponding amino acid.     

The evolution of multi-isoacceptor tRNA families. Sequence of tRNA Leu CAA and tRNA Leu CAG from Anacystis nidulans

Two leucine tRNAs from the cyanophyte Anacystis nidulans have been isolated, and their complete nucleotide sequences have been determined by combining data from oligonucleotide fingerprints and sequencing gels. The two sequences are 87 nucleotides long, have the anticodons CAA and CAG, and differ from each other at a total of 28 positions. They have been compared to other known tRNA Leu sequences and incorporated into a phylogenetic tree comprising prokaryotic and chloroplastic tRNA Leu sequences. Mutations inferred from the tree show that some parts of the tRNA molecule are highly variable (the extra arm and the acceptor stem) while others are much more conserved (the D and T arms). The topology of the tree supports the idea that blue-green algae and chloroplasts share a common prokaryotic ancestor and show a basic divergence between XAA and XAG anticodon-containing tRNAs, suggesting that these two subfamilies result from an ancient gene duplication. Finally, comparison of this phylogenetic tree with those of other multi-isoacceptor tRNA families shows no common scheme, which may be due to independent refinement of codon-reading patterns in different tRNA families.     

Mammalian cells do not have a stringent response

A key attribute of the stringent response of bacteria is the rapid inhibition of ribosomal RNA synthesis mediated by unusual nucleotides in response to uncharged tRNA. The question as to whether mammalian cells show a stringent response analogous to that of bacteria was critically tested by the effective rapid amino acid starvation of both normal and transformed cells. Rapid starvation giving a high proportion of uncharged tRNA for leucine was produced within 7 minutes of expression of a nonleaky ts leucyl tRNA synthetase mutation in transformed CHO cells (tsH1) and in its normal growth control revertant (L-73). To control for the effect of temperature alone, tsrevertants of tsH1 and L-73 were included in the study, and to control for effects due simply to the inhibition of protein synthesis, the translational elongation inhibitor cycloheximide was used. In addition, rapid starvation for histidine was effected by incubation of both the CHO cell lines and of freshly explanted normal Chinese hamster embryo fibroblasts in histidine-free medium containing high concentrations of histidinol. The rate of preribosomal RNA synthesis and the extent of its maturation to mature rRNA was measured using (³H-methyl) methionine as a donor of methyl groups during synthesis and methylation of pre-rRNA. There was no effect on pre-rRNA synthesis of the rapid generation of uncharged tRNA for 45 minutes for any of the cell types tested. A nonspecific inhibition of maturation of 18S rRNA and late (3 hour) inhibition of pre-rRNA synthesis was observed, but could be mimicked by the inhibition of protein synthesis to comparable levels with cycloheximide. Less severe amino acid starvation resulting in a more physiological inhibition of protein synthesis to 30% also had no specific effect on pre-rRNA synthesis and maturation. Intracellular nucleotide pools were also examined for the appearance of unusual nucleotides such as guanosine tetraphosphate or pentaphosphate and for changes in the levels of normal nucleotides after severe amino acid starvation. No such changes could be detected. We conclude that although mammalian cells may have some biochemical reactions which respond to uncharged tRNA, they do not possess a macromolecular control system analogous to the stringent response of bacteria.     

Evolution of acceptor stem tRNA recognition by class II prolyl-tRNA synthetase

Aminoacyl-tRNA synthetases (AARS) are an essential family of enzymes that catalyze the attachment of amino acids to specific tRNAs during translation. Previously, we showed that base-specific recognition of the tRNA(Pro) acceptor stem is critical for recognition by Escherichia coli prolyl-tRNA synthetase (ProRS), but not for human ProRS. To further delineate species-specific differences in acceptor stem recognition, atomic group mutagenesis was used to probe the role of sugar-phosphate backbone interactions in recognition of human tRNA(Pro). Incorporation of site-specific 2'-deoxynucleotides, as well as phosphorothioate and methylphosphonate modifications within the tRNA acceptor stem revealed an extensive network of interactions with specific functional groups proximal to the first base pair and the discriminator base. Backbone functional groups located at the base of the acceptor stem, especially the 2'-hydroxyl of A66, are also critical for aminoacylation catalytic efficiency by human ProRS. Therefore, in contrast to the bacterial system, backbone-specific interactions contribute significantly more to tRNA recognition by the human enzyme than base-specific interactions. Taken together with previous studies, these data show that ProRS-tRNA acceptor stem interactions have co-adapted through evolution from a mechanism involving 'direct readout' of nucleotide bases to one relying primarily on backbone-specific 'indirect readout'.     

Charging levels of four tRNA species in Escherichia coli Rel(+) and Rel(-) strains during amino acid starvation: a simple model for the effect of ppGpp on translational accuracy

Escherichia coli strains mutated in the relA gene lack the ability to produce ppGpp during amino acid starvation. One consequence of this deficiency is a tenfold increase in misincorporation at starved codons compared to the wild-type. Previous work had shown that the charging levels of tRNAs were the same in Rel(+) and Rel(-) strains and reduced, at most, two- to fivefold in both strains during starvation. The present reinvestigation of the charging levels of tRNA(2)(Arg), tRNA(1)(Thr), tRNA(1)(Leu) and tRNA(His) during starvation of isogenic Rel(+) and Rel(-) strains showed that starvation reduced charging levels tenfold to 40-fold. This reduction corresponds much better with the decreased rate of protein synthesis during starvation than that reported earlier. The determination of the charging levels of tRNA(2)(Arg) and tRNA(1)(Thr) during starvation were accurate enough to demonstrate that charging levels were at least fivefold lower in the Rel(-) strain compared to the Rel(+) strain. Together with other data from the literature, these new data suggest a simple model in which mis-incorporation increases as the substrate availability decreases and that ppGpp has no direct effect on enhancing translational accuracy at the ribosome.