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.     

Stability and separation of aminoacyl-transfer RNAs on chromatography columns

In order to demonstrate that the apparent amount of a tRNA isoacceptor depends on the column matrix employed, chromatographic separations of lysyl tRNAs on several polystyrene anion exchangers and on a reversed-phase matrix (RPC-5) have been studied. Experiments were carried out to distinguish between the actual yield of isoacceptors (aminoacylated and free species) and the apparent yield of isoacceptors (aminoacylated species only). The results indicate that several polystyrene anion exchangers with similar physical properties resolve lysyl tRNAs differently. The differences are noted in the apparent yields and in the degree of chromatographic resolution. When fire column matrices are incubated separately with [³H]lysyl tRNAs and the deacylations measured, the results indicate chemical deacylation by two polystyrene anion exchange matrices but not by two other polystyrene matrices or by RPC-5. Further study of two major isoacceptors of lysyl-tRNA on these column matrices confirm that different matrices cause chemical deacylation of the aminoacyl tRNA bond at different rates. Therefore, the apparent yield of an isoacceptor depends upon the column matrix. The dissociation of the ester bond of the aminoacyl tRNA appears to be catalysed by the quaternary ammonium groups of the matrix. Enhanced deesterification of the aminoacyl tRNA bond is noted in slightly alkaline eluants, suggesting a nucleophilic attack by the hydroxyl anion of the medium. The major conclusion to be drawn is that both the yield and relative amounts of isoacceptors are dependent not only upon the resolving power of the column matrix, but also upon the physical and chemical nature of the matrix and upon the experimental conditions employed. This catalytic effect is in addition to the base-catalysed de-esterification promoted by the residual primary, secondary or tertiary amine groups present in the polystyrene anion exchangers.     

The function of the histidine tRNA isoaccepting species in hemoglobin synthesis.

Rabbit reticulocytes contain two RNA isoaccepting species for histidine as resolved by various chromatographic methods, while rabbit liver contains only one. These isoacceptors cannot be distinguished on the basis of coding properties, consistent with the "Wobble Hypothesis" (Smith, D.W.E., Meltzer, V.N., and McNamara, A.L. (1974) Biochim. Biophys. Acta 349, 366-375). Their function in hemoglobin synthesis in reticulocyte lysates has been investigated. Each of the tRNA isoacceptors of reticulocytes and the tRNA species of liver can incorporate histidine into positions in hemoglobin encoded by both of the histidine code words, CAC and CAU, and it is likely that each can incorporate histidine into all of the histidine-containing positions of hemoglobin. Even in experiments in which the two histidine tRNA species of reticulocytes are placed together in a lysate and are therefore in competition with each other, each incorporates histidine into all of the histidine-containing positions. There is no evidence that any residues are incorporated preferentially by either of the tRNA species. The two species are attached to reticulocyte ribosomes in the same proportion as they occur in the reticulocyte, also suggesting that neither of them is used preferentially in hemoglobin synthesis. The first of the two reticulocyte histidine isoacceptors and the histidine tRNA of rabbit liver contain Q base.     

Comparison of Lysyl-Transfer Ribonucleic Acid Species from Vegetative Cells and Spores of Bacillus subtilis by Methylated Albumin-Kieselguhr and Reversed-Phase Chromatography

Lysyl-transfer ribonucleic acid (tRNA) species from a spore-forming strain of Bacillus subtilis (168 trp2(-)) and an early blocked asporogenous mutant (spoA 12) were compared on reversed-phase and methylated albumin-kieselguhr columns. Lysyl-tRNA species from spores and the asporogenous mutant in stationary phase both exhibited altered chromatographic profiles compared to that of log-phase cells. The major peak in spore lysyl-tRNA species eluted later than that characteristic of vegetative cells, whereas the major peak of the lysyl-tRNA species from the asporogenous mutant in stationary phase eluted earlier. Although the early eluting lysyl-tRNA species was observable on methylated albumin columns, the late eluting peak was not detectable by that column technique. By using a shallower gradient on an RPC-2 column, the resolution of all lysyl-tRNA species increased. Several subspecies were revealed. The chromatographic comparisons clearly show that both the spore-forming strain and the asporogenous mutant undergo relative increases in different lysyl-tRNA species when grown to late stationary phase. No new species seem to be involved but rather altered amounts of minor species existing in log-phase cells. The experiments also demonstrate the usefulness of reversed-phase columns for such comparisons.     

Mitochondrial protein-synthesizing machinery in Saccharomyces cerevisiae grown in different metabolic conditions. Variability of seryl-tRNA and alanyl-tRNA isoacceptor patterns.

1. The isoacceptor patterns of mitochondrial seryl and alanyl-tRNAs from Saccharomyces cerevisiae grown under different conditions and committed to fermentative or respiratory metabolism have been studied by reversed-phase chromatography. 2. An extensive variability of the chromatographic patterns of the four isoacceptors for serine and of the three isoacceptors for alanine has been observed as a function of carbon source and stage of growth, but the main differences were observed in the different stages of growth on glucose. 3. In order to distinguish the effects due to stage of growth from those due to relief from glucose repression, the isoacceptor patterns of mitochondrial tRNA were compared in repressed and derepressed resting cells; results show that some of the mitochondrial species are almost undetectable in resting repressed cells, but are the major ones after recovery of respiration. 4. Two of the serine isoacceptors (species 1 and 2), one of which is absent or not acylable in repressed resting cells, are different gene products.     

A preferential role for lysyl-tRNA4 in the synthesis of diadenosine 5',5'-P1,P4-tetraphosphate by an arginyl-tRNA synthetase-lysyl-tRNA synthetase complex from rat liver

The synthesis of diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) can be catalyzed in vitro by a tetrameric tRNA synthetase complex from rat liver containing two lysyl-tRNA synthetase and two arginyl-tRNA synthetase subunits. This reaction required ATP, AMP, 50-100 microM zinc, and inorganic pyrophosphatase. We show here that AMP can be omitted from the reaction and that the zinc levels can be markedly reduced provided catalytic amounts of tRNA(Lys) are added to the reaction mixture. Ap4A synthesis with purified tRNA(Lys) isoacceptors showed that the minor species, tRNA(4Lys), was 3-fold more active than either of the two major tRNA(Lys) species, tRNA(2Lys) and tRNA(5Lys). No activity could be demonstrated with tRNA(Lys) from Escherichia coli or with tRNA(Lys) or tRNA(Phe) from yeast. Aminoacylation of tRNA(4Lys) was strictly required as determined by the fact that Ap4A synthesis was not observed until aminoacylation was nearly complete, inhibitors of aminoacylation blocked Ap4A synthesis, and there was a strict requirement for added lysine. None of the above observations could be demonstrated, however, when lysyl-tRNA(Lys) was directly supplied to the reaction mixture. Optimum Ap4A synthesis was obtained by the addition of 1 mol of tRNA(Lys)/mol of the synthetase complex. This reaction is unique because it does not require the prior formation of an aminoacyl-AMP intermediate and because it can actively synthesize Ap4A at physiological zinc concentrations. The preferential role for tRNA(4Lys) in Ap4A synthesis is consistent with its prior implication in cell division.     

Aminoacyl-tRNAs from Physarum polycephalum: patterns of codon recognition.

Isoacceptors of Physarum polycephalum Ala-, Arg-, Glu-, Gln-, Gly-, Ile-, Leu-, Lys-, Ser-, Thr-, and Val-tRNAs were resolved by reverse-phase chromatography and isolated, and their codon recognition properties were determined in a ribosomal binding assay. Codon assignments were made to most isoacceptors, and they are summarized along with those determined in other studies from Escherichia coli, yeasts, wheat germ, hymenoptera, Xenopus, and mammals. The patterns of codon recognition by isoacceptors from P. polycephalum are more similar to those of animals than to those of plants or lower fungi.     

Dihydrouridine-deficient tRNAs in Saccharomyces cerevisiae.

A mutation in Saccharomyces cerevisiae, designated mia, is responsible for the production of isoaccepting tRNA molecules with reduced extents of nucleoside modifications. The mia isoacceptors of tRNAPhe and one of the mutant isoacceptors of tRNATyr were highly purified for nucleoside composition analyses. The data indicate that the mutant isoacceptors are lacking some of the dihydrouridine moieties. This is consistent with our previous hypothesis that the mutant isoacceptors were accumulated due to a defect in a modification process [Lo, R.Y.C. and Bell, J.B. (1981) Current Genetics 3, 73-82). Data from in vitro poly-U translation experiments also support the previous results, suggesting in vivo biological activity of these mutant tRNAs.     

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.     

Queuosine modification of the wobble base in tRNAHis influences ''in vivo'' decoding properties.

The 'in vivo' decoding properties of four tRNAHis isoacceptors, two from Drosophila melanogaster and two from brewer's yeast, were studied after their microinjection, along with turnip yellow mosaic virus (TYMV) coat protein mRNA, into Xenopus laevis oocytes. The two Drosophila isoacceptors are identical besides containing either a guanosine (G) or the hypermodified nucleoside queuosine (Q) in the wobble position. The brewer's yeast isoacceptors differ by four bases in the anticodon stem, and by one base in the amino acceptor stem. Our results show that, under competing 'in vivo' conditions, the Drosophila tRNAHis with the anticodon GUG clearly prefers the histidine codon CAC to the codon CAU, whereas little preference is observed for the tRNAHis with the anticodon QUG for the codon CAU, and no preference for either codon by the two yeast isoacceptors. Hence, it can be concluded that the presence of the Q-base clearly affects the choice of the codon. This is the first demonstration of an 'in vivo' codon preference by tRNA isoacceptors differing in the modification of the wobble base during the elongation step of protein synthesis. These results imply that one function of the Q-base is at the translational level.     

The interaction between HIV-1 Gag and human lysyl-tRNA synthetase during viral assembly

Human lysyl-tRNA synthetase (LysRS) is a tRNA-binding protein that is selectively packaged into HIV-1 along with its cognate tRNALys isoacceptors. Evidence exists that Gag alone is sufficient for the incorporation of LysRS into virions. Herein, using both in vitro and in vivo methods, we begin to map regions in Gag and LysRS that are required for this interaction. In vitro reactions between wild-type and truncated HIV-1 Gag and human LysRS were monitored using GST-tagged molecules and glutathione-agarose chromatography. Gag/LysRS interaction in vivo was detected in 293FT cells cotransfected with plasmids coding for wild-type or mutant HIV-1 Gag and LysRS, either by monitoring Gag.LysRS complexes immunoprecipitated from cell lysate with anti-LysRS or by measuring the ability of LysRS to be packaged into budded Gag viral-like particles. Based on these studies, we conclude that the Gag/LysRS interaction depends upon Gag sequences within the C-terminal domain of capsid (the last 54 amino acids) and amino acids 208-259 of LysRS. The latter domain includes the class II aminoacyl-tRNA synthetase consensus sequence known as motif 1. Both regions have been implicated in homodimerization of capsid and LysRS, respectively. Sequences falling outside these amino acid stretches can be deleted from either molecule without affecting the Gag/LysRS interaction, further supporting the observation that LysRS is incorporated into Gag viral-like particles independent of its ability to bind tRNALys.     

Isolation and characterization of two 5-fluorouracil-substituted Escherichia coli initiator methionine transfer ribonucleic acids

Escherichia coli initiator methionine tRNA labeled in vivo with 5-fluorouracil (FUra) has been isolated and characterized. The tRNA, with essentially all its uracil and uracil-derived minor bases replaced by FUra, was purified by sequential chromatography, first on diethylaminoethylcellulose (DEAE-cellulose), at pH 8.9, followed by chromatography on Sepharose 4B, using a reverse salt gradient, then on DEAE-Sephadex A-50, and finally on benzoylated DEAE-cellulose. The last step resolved two FUra-substituted tRNAfMet-iso-accepting species, each with a specific activity over 1500 pmol/A260. Kinetic analysis shows both are aminoacylated at the same rate; apparent KmS for the two are 0.92 and 0.94 microM, compared with 1.7 microM for normal tRNAfMet. Chromatographic differences between the two forms of fluorinated tRNAfMet persist after aminoacylation, and the two tRNAs are not interconverted by denaturation and renaturation. The isoacceptors have nearly identical nucleoside composition, and both contain 7-methylguanosine and 2'-O-methylcytidine as the only modified nucleosides. Analysis of complete RNase T1 digests of the two methionine tRNAs shows that they differ in only one oligonucleotide. The sequence 20FpApGp, derived from the dihydrouridine loop and stem region, which is found in one of the isoaccepting forms of the tRNA, is replaced by an oligonucleotide containing adenine and guanine, but no FUra in the other. A modified FUra, with the properties of a 5-fluoro-5,6-dihydrouracil derivative, is detected in this tRNA. 19F NMR spectra of the two species of FUra-substituted initiator tRNA show 9-10 resolved resonances for the 12 FUra residues incorporated. The spectra differ primarily in the shift of one peak in the form lacking the sequence 20FpApGp, from 4.8 ppm downfield from free FUra (= 0 ppm) to 14.9 ppm upfield from the standard.     

Specific changes in Q-ribonucleoside containing transfer RNA species during Friend leukemia cell erythroid differentiation.

Changes in specific tRNA isoacceptors during Friend leukemia cell (F.L.C.) erythroid differentiation have been found to be concomitant with differences in the extent of the Q-base modification in certain species of tRNA. Transfer RNA was isolated from F.L.C. cultures after 0, 36, 48, 72, and 96 hr of DMSO induced differentiation. Changes in 17 isoacceptors of tRNAasn, tRNAasp, tRNAhis and tRNAtyr were compared by RPC-5 chromatography. Isoacceptors of these tRNA changed in relative amounts, following consistent trends throughout cell differentiation. The amount and distribution of Q-base containing tRNA isoacceptors was assayed by measuring the quanine-tRNA transferase catalyzed incorporation of [3H]-labeled guanine into tRNA species undermodified in Q-base followed by RPC-5 chormatography of the tRNA. The amount of Q-base containing tRNA species decreased in the first 48 hr after the induction, then increased again, indicating the level of Q-modification is correlated to the process of differentiation. Isoacceptors that lacked the Q-base were eluted late from RPC-5.