Over expression of a tRNA(Leu) isoacceptor changes charging pattern of leucine tRNAs and reveals new codon reading

During mRNA translation, synonymous codons for one amino acid are often read by different isoaccepting tRNAs. The theory of selective tRNA charging predicts greatly varying percentages of aminoacylation among isoacceptors in cells starved for their common amino acid. It also predicts major changes in tRNA charging patterns upon concentration changes of single isoacceptors, which suggests a novel type of translational control of gene expression. We therefore tested the theory by measuring with Northern blots the charging of Leu-tRNAs in Escherichia coli under Leu limitation in response to over expression of tRNA(GAG)(Leu). As predicted, the charged level of tRNA(GAG)(Leu) increased and the charged levels of four other Leu isoacceptors decreased or remained unchanged, but the charged level of tRNA(UAG)(Leu) increased unexpectedly. To remove this apparent inconsistency between theory and experiment we postulated a previously unknown common codon for tRNA(GAG)(Leu) and tRNA(UAG)(Leu). Subsequently, we demonstrated that the tRNA(GAG)(Leu) codon CUU is, in fact, read also by tRNA(UAG)(Leu), due to a uridine-5-oxyacetic acid modification.     

Relationships Among Deoxyribonucleic Acid, Ribonucleic Acid, and Specific Transfer Ribonucleic Acids in Escherichia coli 15T− at Various Growth Rates

The levels of macromolecules in Escherichia coli 15T(-) growing in broth, glucose, succinate, and acetate media were determined to compare relationships among deoxyribonucleic acid (DNA), ribosomal ribonucleic acid (rRNA), transfer RNA (tRNA), and protein in cells at different growth rates. DNA and protein increased in relative amounts with decreasing growth rate; relative amounts of rRNA and tRNA decreased, tRNA making up a slightly larger proportion of RNA. For several amino acid-specific tRNAs studied, acceptor capacities per unit of DNA increased with increasing growth rate. The syntheses of tRNA and rRNA are regulated by similar, yet different, mechanisms. Chromatographic examination on columns of benzoylated diethylaminoethyl-cellulose of isoaccepting tRNAs for arginine, leucine, lysine, methionine, phenylalanine, serine, and valine did not reveal differences in the isoaccepting profiles for rapidly (broth culture) and slowly growing (acetate culture) cells. Therefore, isoacceptors for individual amino acids appear to be regulated as a group. Lower efficiencies of ribosomal function in protein synthesis can be explained, in part, by a low ratio of tRNA to the number of ribosomes available and by a decreasing concentration of tRNA with decreasing growth rate. Data on the tRNAs specific for seven amino acids indicate that the decreasing concentration of tRNA is a general event rather than a severe limitation of any one tRNA or isoaccepting tRNA.     

Alterations in tRNA isoaccepting species during erythroid differentiation of the Friend leukemia cell.

The chromatographic profiles of isoaccepting tRNAs were analyzed at five time points during the 96 hr, dimethylsulfoxide induced, erythroid-like differentiation of Friend leukemia cells. Sixty-four isoaccepting species of tRNA for 16 amino acids were resolved by RPC-5 chromatography. The relative amounts of tRNAphe, tRNAile, and tRNAval species were maintained by the cells during differentiation; whereas the relative amounts of some of the isoacceptor tRNAs for the other 13 amino acids changed significantly. Fluctuations in amounts of isoacceptors occurred between 36 and 72 hr after addition of dimethysulfoxide, corresponding to globin mRNA appearance and hemoglobin synthesis, respectively. In most cases, thepredominant tRNA isoacceptors of uninduced cells were retained throughout differentiation. Notable exceptions were tRNA species for threonine, proline, and methionine. Some of the isoacceptors occurring in relatively smaller amounts were not expressed at all times. These changes possibly reflect the cell's functional adaptation of tRNA in differentiation for hemoglobin synthesis.     

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.     

Genetically determined differences in concentrations of isoaccepting tRNAs in Escherichia coli.

Two examples of genetically determined altered concentrations of isoaccepting tRNAs are presented. The concentrations of isoaccepting tRNAsThr are selectively changed by a mutation causing a fourfold overproduction of the cognate aminoacyl-tRNA-synthetase, the threonyl-tRNA synthetase, whereas the distribution of isoaccepting tRNAs of four control tRNA-species in these E. coli mutants was not affected by that mutation. Secondly evidence is presented for a correlation between mutations in structural genes of aminoacid biosynthetic enzymes and alterations in concentrations of cognate isoaccepting tRNAs in two different E. coli strains, auxotrophic for threonine, isoleucine/valine and leucine, and arginine respectively.     

Chromatographic Analyses of Isoaccepting tRNAs from Avian Myeloblastosis Virus

Avian myeloblastosis virus (AMV) 4S RNA was tested for amino acid acceptor activity for 18 of the 20 amino acids. A nonrandom distribution of viral tRNAs was found compared with tRNA from normal liver or from AMV-infected leukemic myeloblasts, confirming previous reports. Methionine and proline tRNAs were considerably enriched, whereas glutamic acid, glutamine, serine, tyrosine, and valine tRNAs were markedly depleted in AMV relative to homologous cellular tRNAs. The seven AMV tRNAs with the greatest amino acid acceptance capacities, which were in order methionine, proline, lysine, arginine, histidine, isoleucine, and threonine tRNAs, were compared with homologous tRNAs from leukemic myeloblasts and liver by reversed-phase 5 chromatography. Of the 25 isoaccepting chromatographic fractions identified, no tRNA species unique to AMV was detected. Only methionyl-tRNA showed a substantial quantitative variation in isoaccepting species compared with the host cell. Thus, viral selectivity for amino acid-specific tRNAs is not, generally, paralleled by selectivity for individual isoaccepting tRNA species. Qualitative differences in arginyl- and histidyl-tRNA isoaccepting species were discovered in virus and leukemic myeloblasts compared with liver. This indicates the existence of structural differences in these tRNA species which could be related to virus replication or expression.     

Adaptation of the tRNA population of maize endosperm for zein synthesis.

Maize endosperm, 30 days after pollination is actively synthesizing zein, a storage protein containing high amounts of glutamine. leucine and alanine. Endosperm tRNAs have a higher accepting activity than embryo tRNAs for these three amino acids, but not for some other (control) amino acids. This increase in accepting activity is accompanied by a change in the distribution of the isoaccepting tRNA species corresponding to these three amino acids, but not of the isoacceptors corresponding to some other (control) amino acids. These results are in favor of the theory of functional adaptation of tRNA population.     

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.     

RNA minihelices and the decoding of genetic information

The rules of the genetic code are determined by the specific aminoacylation of transfer RNAs by aminoacyl transfer RNA synthetase. A straightforward analysis shows that a system of synthetase-tRNA interactions that relies on anticodons for specificity could, in principle, enable most synthetases to distinguish their cognate tRNA isoacceptors from all others. Although the anticodons of some tRNAs are recognition sites for the cognate aminoacyl tRNA synthetases, for other synthetases the anticodon is dispensable for specific aminoacylation. In particular, alanine and histidine tRNA synthetases aminoacylate small RNA minihelices that reconstruct the part of their cognate tRNAs that is proximate to the amino acid attachment site. Helices with as few as six base pairs can be efficiently aminoacylated. The specificity of aminoacylation is determined by a few nucleotides and can be converted from one amino acid to another by the change of only a few nucleotides. These findings suggest that, for a subgroup of the synthetases, there is a distinct code in the acceptor helix of transfer RNAs that determines aminoacylation specificity.     

Transfer RNATyr of melanoma tissues and cells: relevance to melanin synthesis?

The tRNA present in swine melanoma tumor tissue and normal gray skin tissue were compared by aminoacylation of the unfractionated tRNA preparations. Of the seventeen amino acids studied, seven showed differences in rate of acceptance to tRNAs from normal and tumor tissues; the tRNAs of two amino acids, tyrosine and glycine, showed dramatic three fold increases in melanoma tumor. As melanin biosynthesis proceeds from tyrosine oxidation the investigations focused on the increase in tyrosine tRNA. Kinetic analysis of tyrosine aminoacylation to normal and melanoma tRNAs revealed no differences. Analysis of the isoaccepting species of tRNATyr from normal skin and melanoma tumor tissues identified three isoacceptors; tRNATyr, represented the predominant species in normal gray skin, while tRNA2Tyr predominated in melanoma tumor tissue. The tyrosine acceptances by tRNAs from three human melanoma cell lines were analyzed and found to be variable, but isoaccepting species analysis of the tRNATyr of these three cell lines still showed a correlation between the preponderance of tRNA2Tyr and extent of tyrosine acceptance. Additionally the enzymatic activity for the oxidation of tyrosine was found to be related to tyrosine acceptance and tRNA2Tyr predominance..     

Response of specific transfer-ribonucleic-acid levels to amino-acid deprivation in Friend leukemia cells.

In eukaryotes, the levels of specific tRNAs are closely correlated with the demands for their cognate amino acids in protein synthesis. To account for this phenomenon, we have proposed that the extent of aminoacylation of a given tRNA species in vivo controls the relative rate of synthesis or turnover of that species. Previously, we reported that Friend leukemia cells respond to histidine deprivation by increasing their relative level of tRNAHis by as much as two-fold, with no change in the relative level of tRNALeu. In this paper, we show that deprivation of leucine or tryptophan also causes a specific increase in the relative level of tRNAs cognate to the deprived amino acid. At least in the case of tRNATrp, the increases in relative tRNA levels are preceded by extensive declines in the steady-state extent of aminoacylation of the tRNA in vitro. We also find that different isoacceptors may respond differently to amino acid deprivation. These results suggest that decreased extents of aminoacylation of a given tRNA species in vivo cause increases in the relative rate of synthesis or decreases in the relative rate of degradation of that species.     

Changes in Transfer Ribonucleic Acids Accompanying Encystment in Acanthamoeba castellanii

Transfer ribonucleic acid (tRNA) from exponentially growing cells (trophozoites) and from precysts of Acanthamoeba castellanii were examined by reversed-phase column (RPC-2) chromatography. This system gave excellent resolution of isoaccepting species of tRNA. The tRNAs for 12 amino acids were studied. A comparison of trophozoite and precyst tRNA elution profiles revealed no apparent differences in the number of isoaccepting species of alanyl-, arginyl-, asparaginyl-, glycyl-, leucyl-, lysyl-, methionyl-, phenylalanyl-, tryptophanyl-, or valyl-tRNAs. Seryl-tRNAs from trophozoites were eluted as three components, whereas precyst seryl-tRNAs were eluted as only two components. Precharged trophozoite and precyst isoleucyl-tRNAs were both eluted as single components; however, post-chromatography charging of trophozoite tRNA resulted in three components of activity for tRNA(Ile) and only one component for precyst tRNA(Ile). None of the observed changes could be attributed to differences in synthetases or to the presence of altered tRNA lacking the CCA terminus or partially degraded by nucleases. The possible significance of these observations is discussed.     

Purification of Leucine tRNA Isoaccepting Species from Soybean Cotyledons: II. RPC-2 Purification, Ribosome Binding, and Cytokinin Content

Two of the six leucine isoaccepting tRNA species from soybean (Glycine max) cotyledons recognize U-beginning codons, and contain cytokinin moieties in their structure. These same two isoaccepting species have been shown to undergo quantitative changes in their relative amounts upon treatment with N⁶-benzyladenine in vivo. In addition a procedure has been developed for purification of the isoaccepting species of leucine tRNA from soybean cotyledons resulting in isoacceptors of minimum purity, calculated by amino acid acceptance capacity, of from 46 to 78% leucine tRNA.     

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.     

Multiple isoacceptor forms of several transfer ribonucleic acids in a mutant yeast strain.

By use of reverse phase 5 chromatography, a strain of Saccharomyces cerevisiae (XB 109-5B) has been shown to exhibit multiple isoaccepting forms for several of the transfer ribonucleic acids (tRNAs). This is in contrast with a standard wild-type strain where only one acceptor is found for each tRNA studied. Multiple peaks for tRNATyr, tRNAPhe, tRNASer, and tRNAVal have been detected for strain XB 109-5B. However, the observation of multiple isoacceptors cannot be extended to all tRNAs in this strain since tRNAAsp appears as a single form that is the same as in the wild type. The appearance of multiple peaks was found to depend on the growth conditions of the cells. The tRNA profiles of XB 109-5B that was grown rapidly with vigorous aeration differed the most from profiles of comparably grown wild-type yeast, whereas tRNA from this mutant, grown without shaking or supplementary aeration, appeared the same as the wild type. The minor nucleoside composition of the isoacceptors of tRNAPhe was obtained.     

Functional idiosyncrasies of tRNA isoacceptors in cognate and noncognate aminoacylation systems

The specificity of transfer RNA aminoacylation by cognate aminoacyl-tRNA synthetase is a crucial step for synthesis of functional proteins. It is established that the aminoacylation identity of a single tRNA or of a family of tRNA isoacceptors is linked to the presence of positive signals (determinants) allowing recognition by cognate synthetases and negative signals (antideterminants) leading to rejection by the noncognate ones. The completion of identity sets was generally tested by transplantation of the corresponding nucleotides into one or several host tRNAs which acquire as a consequence the new aminoacylation specificities. Such transplantation experiments were also useful to detect peculiar structural refinements required for optimal expression of a given aminoacylation identity set within a host tRNA. This study explores expression of the defined yeast aspartate identity set into different tRNA scaffolds of a same specificity, namely the four yeast tRNA(Arg) isoacceptors. The goal was to investigate whether expression of the new identity is similar due to the unique specificity of the host tRNAs or whether it is differently expressed due to their peculiar sequences and structural features. In vitro transcribed native tRNA(Arg) isoacceptors and variants bearing the aspartate identity elements were prepared and their aminoacylation properties established. The four wild-type isoacceptors are active in arginylation with catalytic efficiencies in a 20-fold range and are inactive in aspartylation. While transplanted tRNA(1)(Arg) and tRNA(4)(Arg) are converted into highly efficient substrates for yeast aspartyl-tRNA synthetase, transplanted tRNA(2)(Arg) and tRNA(3)(Arg) remain poorly aspartylated. Search for antideterminants in these two tRNAs reveals idiosyncratic features. Conversion of the single base-pair C6-G67 into G6-C67, the pair present in tRNA(Asp), allows full expression of the aspartate identity in the transplanted tRNA(2)(Arg), but not in tRNA(3)(Arg). It is concluded that the different isoacceptor tRNAs protect themselves from misaminoacylation by idiosyncratic pathways of antidetermination.     

Levels of tRNAs in bacterial cells as affected by amino acid usage in proteins.

Transfer RNAs of Mycoplasma capricolum were separated by two-dimensional polyacrylamide gel electrophoresis, and the relative abundance of each of the 28 known tRNA species was measured. There existed a correlation between the relative amount of isoacceptor tRNAs and the frequency in choosing synonymous codons that could be translated by the isoacceptors. Furthermore, it was observed that the total amount of tRNAs for a particular amino acid was paralleled by the composition of the amino acid in ribosomal proteins. A similar relationship was obtained from reexamination of the previous data on Escherichia coli tRNAs, suggesting that the amount of tRNAs for an amino acid is affected by the usage of the amino acid in proteins.     

Amino acid modifications on tRNA

The accurate formation of cognate aminoacyl-transfer RNAs (aa-tRNAs) is essential for the fidelity of translation. Most amino acids are esterified onto their cognate tRNA isoacceptors directly by aa-tRNA synthetases. However, in the case of four amino acids (Gln, Asn, Cys and Sec), aminoacyl-tRNAs are made through indirect pathways in many organisms across all three domains of life. The process begins with the charging of noncognate amino acids to tRNAs by a specialized synthetase in the case of Cys-tRNA(Cys) formation or by synthetases with relaxed specificity, such as the non-discriminating glutamyl-tRNA, non-discriminating aspartyl-tRNA and seryl-tRNA synthetases. The resulting misacylated tRNAs are then converted to cognate pairs through transformation of the amino acids on the tRNA, which is catalyzed by a group of tRNA-dependent modifying enzymes, such as tRNA-dependent amidotransferases, Sep-tRNA:Cys-tRNA synthase, O-phosphoseryl-tRNA kinase and Sep-tRNA:Sec-tRNA synthase. The majority of these indirect pathways are widely spread in all domains of life and thought to be part of the evolutionary process.