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.     

Preferential usage of tRNA isoaccepting species in collagen synthesis.

A new double label technique is described for determining the usage of individual isoaccepting tRNA species by in vitro protein-synthesizing systems. Employing this method we show that of four major species of glycyl-tRNA one which is cognate to GGU and GGC is used predominantly in collagen synthesis by polysomes isolated from embryonic chick calvaria. A similar preferential usage was found for one of two alanyl-tRNA species. No preference for any particular isoaccepting tRNA species was observed in the synthesis of noncollagenous proteins by either calvaria or liver polysomes except for the disproportionate usage of one lysyl-tRNA species. Whether such preferential usage permits translational control of collagen synthesis in vivo remains to be determined.     

Alteration of the intracellular concentration of aminoacyl-tRNA synthetases and isoaccepting tRNAs during amino-acid limited growth in Escherichia coli.

Growth of Escherichia coli AB 2271 under threonine or isoleucine deficiency leads to a depression of the threonyl-tRNA synthetase and isoleucyl-tRNA synthetase respectively. During this amino-acid-limited growth the concentrations of isoaccepting fractions of the cognate tRNA species were changed, as demonstrated by their altered reversed-phase-5 chromatograms. But, in addition, the profiles of the isoacceptors of all other tRNA species investigated, i.e. of tRNAsLeu, tRNAsSer and tRNAsArg were also altered. This means that, if there is a correlation between regulation of the level of an aminoacyl-tRNA synthetase and its cognate isoaccepting tRNAs, it is superimposed by the effect of amino acid limitation upon the concentration of all isoaccepting tRNAs. So far drastic changes in profiles of isoaccepting tRNAs have only been observed under unbalanced growth in relaxed cells or during treatment with antibiotics. Here we demonstrate that similar heavy alterations in patterns of isoaccepting tRNAs occur in a proven stringent E. coli strain growing exponentially under amino acid limitation. Thus the observed changes in the profiles of isoaccepting tRNAs during amino acid limitation signal a meaningful biological function of those newly or increasingly occurring isoaccepting tRNAs. During the growth under amino acid limitation the total acceptor activity of eight investigated tRNA species, however, stayed unchanged, except that under threonine-limited growth the total amount of tRNAIle was reduced to about half and that of tRNAGlu increased; both tRNA species of these isoacceptors are known [30,31] as spacers between ribosomal RNAs.     

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..     

Preferential usage of glycyl-tRNA isoaccepting species in collagen synthesis.

Chick embryo tRNA charged with [3H]glycine was incubated in an in vitro protein-synthesizing system using polysomes isolated from either chick embryo liver or calvaria. Using collagenase digestion to measure the fraction of protein synthesized which was collagenous, the results indicate that in the calvaria system approximately 65% of the incorporated [3H]glycine was in collagen. The incorporation of [3H]glycine into protein from individual isoaccepting species was determined by chromatography on a reversed phase system of the charged tRNA before and after incubation in the polysome systems. In the calvaria system, a single tRNAGly species cognate to GGU and GGC and which is found in unusually large amounts in collagen-synthesizing tissues was used preferentially in collagen-synthesizing tissues was used preferentially in collagen synthesis. In the liver system, the rate of incorporation was similar to the calvaria, but no collagen synthesis was detected and only a relatively small preferential usage of any of the four major isoaccepting species was observed. These results support the notion that the complement of tRNA found in a cell may be adapted to the synthesis of a particular protein. It is also possible that under certain circumstances, collagen synthesis may be controlled in vivo at the translational level by the concentration of particular tRNA species.     

The use of a dipolar ion-exchanger for the fractionation of transfer ribonucleic acid.

Transfer ribonucleic acid is well fractionated on columns of arginine-agarose, whose properties appear in general to be similar to those of DEAE-Sephadex. However, the amino acid acceptor species are separated into sharper peaks and in several instances, notably for methionine, glycine, serine, leucine and aspartate accepting tRNAs from Escherichia coli, isoaccepting species are well resolved. In the case of methionine accepting tRNA from E. coli the tRNA Met-m species is eluted before the tRNA Met-f species and since it is also eluted prior to the bulk of the tRNA it is obtained in a high degree of purity. By comparing the properties of columns of arginine-agarose and its methyl ester in which the carboxylate anion is blocked, it is seen that the carboxylate ion plays a role in the fractionation of the tRNA Met species.     

A mutation in the small (alpha) subunit of glycyl-tRNA synthetase affects amino acid activation and subunit association parameters

A strategy was designed to isolate mutants of glycyl-tRNA synthetase that are altered at the amino acid binding site, including a class with altered amino acid specificity. For this purpose, the plasmid pBR322 was mutated so that the codon (AGC) of the active site Ser-68 in the beta-lactamase gene was changed to the glycine codon GGC to inactivate the encoded enzyme. Suppressors that increase the amount of beta-lactamase activity of the Gly-68 allele of beta-lactamase were isolated and some mapped to the gene encoding glycyl-tRNA synthetase (glyS). While in vitro misaminoacylation of tRNA(Gly) with serine was not detected for any of the mutants, glycyl-tRNA synthetase activity was altered. One severely affected glyS mutant (N302) was studied in more detail. For this mutant, a single Pro-61----Leu substitution in the alpha chain confers an elevation of the Km values for glycine (25-fold) and for ATP (45-fold) in the aminoacylation reaction, but only a minor perturbation of the Km for tRNA. There also was a severely reduced adenylate synthesis activity (greater than 100-fold). In addition, a nonlinear dependence between aminoacylation activity and enzyme concentration was observed which implies that the alpha chain Pro-61----Leu mutation has disrupted the functionally essential subunit interactions of the holoenzyme. The results of the preceding paper have shown that the alpha chain and parts of the beta chain are required for aminoacylation and adenylate synthesis activity. The results of this study suggest that the alpha chain specifically contributes to amino acid and to ATP binding in a way that is affected by proper subunit interactions.     

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.     

Subunit structure and tRNA-binding properties of Bombyx mori Glycyl-tRNA synthetase

Large amounts of glycyl-tRNA synthetase were purified from the posterior silk glands of Bombyx mori. The synthetase was estimated to be a dimer with a molecular weight of 180,000. When the enzyme solution was diluted, the dimer dissociated into monomers which were inactive in tRNA aminoacylation. The aminoacylation was investigated with two isoaccepting tRNAsGly isolated from the posterior silk glands. Transfer RNA1Gly was aminoacylated 2-fold faster than tRNA2Gly. Transfer RNA-binding experiments revealed that tRNA1Gly binds with the enzyme in a molar ratio of 2:1, whereas tRNA2Gly formed a 1:1 complex with the enzyme. Based on these experimental results, we proposed that the Bombyx mori glycyl-tRNA synthetase has two active sites for tRNA aminoacylation and that the number of tRNA molecules bound on the synthetase closely correlates with the velocity of aminoacylation.     

Nucleotide sequence studies of normal and genetically altered glycine transfer ribonucleic acids from Escherichia coli.

The total nucleotide sequence of tRNAGGA/G -Gly2 from Escherichia coli is pG-C-G-G-G-C-A-U-C-G-U-A-U-A-A-U-G-G-C-U-A-U-U-A-C-C-U-C-A-G-C-C-U-N-C-C-A-A-G-C-U-G-A-U-G-A-U-G-C-G-G-G-T-psi-C-G-A-U-U-C-C-C-G-C-U-G-C-C-C-G-C-U-C-C-AOH, where T- at position 53 is ribothymidylic acid, and psi- at position 54 is pseudouridylic acid; N- at position 36 is an unidentified derivative of uridylic acid, and is present in modified form in a portion of tRNAGGA/G -Gly 2 molecules isolated from E. coli cells. The missense suppressor mutation, glyTsuA36(HA), results in a C yields U base substitution at the 3' end of the anticodon of tRNAGGA/G -Gly 2 (nucleotide position 38). A secondary effect of this base substitution is the modification of the A residue directly adjacent to the 3' end of the anticodon of tRNAsuA36(HA), -Gly 2 suggesting that the enzymes responsible for this modification recognize the anticodon sequences of prospective tRNA substrates. The creation of a missense-suppressing tRNA, tRNAsuA36(HA), -Gly 2 by an alteration of the anticodon sequence of tRNAGGA/G -Gly 2 is analogous to mechanisms whereby other suppressor tRNAs have arisen. The high degree of nucleotide sequence homology between the amino acid acceptor stems and anticodon regions of four glycine isoaccepting tRNAs specified by E. coli and bacteriophage T4 suggests that these regions may be recognized by the glycyl-tRNA synthetase; the involvement of the anticodon region in the synthetase recognition process is supported by the greatly decreased rate of aminoacylation of tRNAsuA36(HA) -Gly 2.     

Characterization of a proteolipid complex of aminoacyl-tRNA synthetases and transfer RNA from rat liver.

A high molecular weight complex containing aminoacyl-tRNA synthetases, peptidyl acetyltransferase, lipids and tRNA has been isolated from the 250,000 x g postmitochondrial supernatant from rat liver cells. Aminoacyl-tRNA synthetase activity directed towards arginine, aspartate, glutamine, glutamate, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and tyrosine is present. An endogenous pool of aminoacyladenylates is indicated by an ATP-32PPi exchange catalyzed by the native complex, which shows a dramatic increase after addition of ATP. Lysine is the only amino acid which greatly increases the exchange rate catalyzed by the native complex in vitro, whereas components of the denatured complex activate all the 13 amino acids in the presence of ATP. Six of the eight lipid fractions were glycolipids; cholesterol and cholesterol esters were absent. The extracted RNA has many characteristics of tRNA. These findings provide evidence for the organization of aminoacyl-tRNA synthetases in a complex with peptidyl acetyltransferase that also contains lipids and tRNA and that can be readily isolated from the cytosol of rat liver cells.     

Evidence that translational control mechanisms operate to optimize antifreeze protein production in the winter flounder

In fall and winter, the liver of the winter flounder produces large amounts of alanine-rich (60 mol %) antifreeze proteins for export to the circulation. We have examined the tRNA in the liver to see if the seasonal production of antifreeze protein is accompanied by changes in tRNAAla isoacceptors. Total tRNA from the liver of winter fish showed an approximate 40% increase in alanine acceptor capacity over tRNA from summer fish. In contrast, the acceptor capacities for other amino acids showed no seasonal difference. When labeled alanyl-tRNAs were separated by reverse phase chromatography-5 chromatography, a large proportion of the increase in alanine acceptor capacity was in one of three main peaks. Measurements of the optimum temperatures for various flounder amino-acyl-tRNA synthetases suggest that alanyl-tRNA synthetase functions best between 0 and 5 degrees C, which is the sea water temperature when antifreeze protein synthesis occurs, while prolyl- and valyl-tRNA synthetases are most active between 20 and 30 degrees C. These differences in temperature optima and the seasonal variation in tRNAAla levels and isoaccepting species may both serve to optimize antifreeze protein production by increasing the translational efficiency of its mRNA.     

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.     

The biosynthesis of transfer RNA in insects. I. Increase of amino acid acceptor activity of specific tRNA's utilized for silk protein biosynthesis in the silk gland of Bombyx mori.

1) To detect the quantitative changes of amino acid acceptor activity of tRNA's from the posterior and middle silk glands of Bombyx mori at various ages, a relatively simple and rapid method was established using a mixture of radioactive amino acids in Chlorella hydrolysate. 2) The acceptor activities of silk gland tRNA for 15 amino acids tested seemed to be almost on the same level at the end of the 4th moult stage. During the 5th instar, however, characteristic increases were observed in glycine, alanine, and serine acceptor activities in both silk glands. 3) In the posterior silk gland, which produces fibroin, the acceptor activities for glycine and alanine increased more than that for serine. In the middle silk gland, which produces sericine, the acceptor activity for serine increased more than those for glycine and alanine. 4) In the light of observations on the increase of corresponding aminoacyl-tRNA synthetase activities in the silk glands, a functional adaptation of tRNA synthesis in the tissue is discussed.     

Comparative X-ray structure analysis of human and Escherichia coli tRNA(Gly) acceptor stem microhelices

tRNA identity elements assure the correct aminoacylation of tRNAs by the aminoacyl-tRNA synthetases with the cognate amino acid. The tRNA^Gly/glycyl-tRNA sythetase system is member of the so-called ‘class II system’ in which the tRNA determinants consist of rather simple elements. These are mostly located in the tRNA acceptor stem and in the glycine case additionally the discriminator base at position 73 is required. Within the glycine-tRNA synthetases, the archaebacterial/human and the eubacterial sytems differ with respect to their protein structures and the required tRNA identity elements, suggesting a unique evolutionary divergence.In this study, we present a comparison between the crystal structures of the eubacterial Escherichia coli and the human tRNA^Gly acceptor stem microhelices and their surrounding hydration patterns.     

Transfer RNA of a collagen producing tissue,chick-embryo calvaria

Transfer RNa was isolated from calvaria prepared from chick embryos incubated for 15–17 days. The chargeability of the unfractionated tRNA with ten amino acids tested was very similar to that of unfractionated tRNA from adult chicken liver when data were expressed on the basis of pmoles of amino acceptance per A₂₆₀ unit of tRNA. However, the relative amount of tRNA in calvaria is only about one-fourth the amount in liver. Analysis of individual species of tRNA by two-dimensional electrophoresis on polyacrylamide gels shows that there are fewer isoaccepting species of tRNA in calvaria than in liver.     

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.     

Study of the transfer RNAs coded by T2, T4, and T6 bacteriophages.

T2, T4, and T6 bacteriophage tRNAs coding for arginine, leucine, proline, isoleucine, and glycine were isolated under conditions of short term and long term infection of Escherichia coli B cells. The corresponding phage tRNA species were examined for sequence homology by RNA-DNA hybridization analysis and by their relative behavior on reversed phase chromatography. The results indicate that all three T-even phages code for similar tRNA species; however, some tRNA species are homologous, others are not, and not all of the same tRNA species are coded by each bacteriophage. Reversed phase chromatography showed the presence of isoacceptor tRNAs for each phage aminoacyl-tRNA species. Pulse-chase experiments for [32P]tRNAGly suggest that the multiple isoacceptor species observed derive from the intracellular modification of a single tRNAGly gene product.