Chromatographic Analyses of Isoaccepting tRNAs from Avian Tumor Viruses

Low-molecular-weight RNA from transforming viruses (Rous sarcoma virus-Rous-associated virus 1, Schmidt-Ruppin strain of Rous sarcoma virus, and sarcoma-B(77)), from nontransforming viruses (Rous-associated virus 1 and sarcoma-NTB(77)), and from chicken liver, chicken embryo fibroblast, and Rous sarcoma virus-Rous-associated virus 1-transformed chicken embryo fibroblast was isolated and purified. To determine if there are modified, qualitatively or quantitatively different isoaccepting species of tRNA in these avian sarcoma viruses as compared with the cell of virus origin, chicken embryo fibroblast or normal chicken liver, methionyl-, arginyl-, and lysyl-tRNA (with high amino acid acceptance activity), and aspartyl- and glutamyl-tRNA from viral-trans-formed cells (with low viral amino acid acceptance activity) were co-chromatographed on reversed phase-5 chromatography columns, and elution profiles were compared. Although in each case the elution profile between a particular viral and host cell tRNA differed quantitatively, there was no qualitative difference in the profiles of corresponding tRNAs from either transforming or nontransforming viruses examined. Minor quantitative differences in the elution profiles might be a reflection of the metabolic state of the cells, since all evidence points to acceptor activity being of host rather than viral origin. Since, with the exception of selective packaging of methionyl-tRNA (IV) species by both transforming and nontransforming viruses, no selectivity was found for isoacceptor species of other tRNAs, it seems that such preferential packaging of methionyl-tRNA (IV) species has no bearing on the event of viral transformation.     

Acceptor activity, isoacceptor profiles and function in protein synthesis of transfer RNAs from regenerating skeletal muscle

Transfer RNAs have been prepared from control and regenerating rat skeletal muscle. The yield of tRNA is highest during the early stages of the regeneration process (5 and 8 days following the induction of regeneration) and decreases to near control values thereafter. The amino acid acceptor activity (extent of aminoacylation) of tRNA from regenerating muscle was also found to be higher for some amino acids than the activity of control tRNA, and the maximum increase in activity was observed between 5 and 8 days following the initiation of regeneration with a decrease to control levels through 15 and 30 days. The isoacceptor pattern, determined by RPC-5 chromatography, for methionyl-tRNAs from control muscle and 5-day regenerating muscle were essentially indistinguishable, while a minor peak of prolyl-tRNA was observed in the population from 5-, 8- and 15-day regenerates which was apparently absent from the control tRNA. Lysyl-tRNAs from control muscle contain two major isoacceptors while a third isoacceptor is observed in the tRNA preparations from 5-, 8- and 15-day regenerating muscle. The relative amount of this third isoacceptor is highest in the 8-day population and decreases in amount in tRNAs from 15- and 30-day regenerates. Control muscle also contains two major glutamyl-tRNA species while a third isoacceptor can be detected in regenerates. The relative amount of this species increases during the early course of the regeneration process but is present at near control levels by 30 days following Marcaine injection. Cell-free protein synthesis using muscle polyribosomes showed that tRNAs from regenerating muscle were more effective in stimulating [³⁵S]methionine incorporation than tRNAs from control muscle.     

tRNA over-expression in breast cancer and functional consequences

Increased proliferation and elevated levels of protein synthesis are characteristics of transformed and tumor cells. Though components of the translation machinery are often misregulated in cancers, what role tRNA plays in cancer cells has not been explored. We compare genome-wide tRNA expression in cancer-derived versus non-cancer-derived breast cell lines, as well as tRNA expression in breast tumors versus normal breast tissues. In cancer-derived versus non-cancer-derived cell lines, nuclear-encoded tRNAs increase by up to 3-fold and mitochondrial-encoded tRNAs increase by up to 5-fold. In tumors versus normal breast tissues, both nuclear- and mitochondrial-encoded tRNAs increase up to 10-fold. This tRNA over-expression is selective and coordinates with the properties of cognate amino acids. Nuclear- and mitochondrial-encoded tRNAs exhibit distinct expression patterns, indicating that tRNAs can be used as biomarkers for breast cancer. We also performed association analysis for codon usage-tRNA expression for the cell lines. tRNA isoacceptor expression levels are not geared towards optimal translation of house-keeping or cell line specific genes. Instead, tRNA isoacceptor expression levels may favor the translation of cancer-related genes having regulatory roles. Our results suggest a functional consequence of tRNA over-expression in tumor cells. tRNA isoacceptor over-expression may increase the translational efficiency of genes relevant to cancer development and progression.     

Synthesis of elastin and procallagen by cells from embryonic aorta. Differences in the role of hydroxyproline and the effects of proline analogs on the secretion of the two proteins.

Transfer ribonucleic acid (tRNA) profiles in human lymphocytes stimulated by various mitogens have been compared with profiles from nonstimulated cells and from leukemic cells using reversed-phase chromatography. Comparisons of [³H]- or [¹⁴C]uridine- or [³²P]phosphate-labeled tRNAs showed that the greatest changes in tRNA composition upon phytohemagglutinin (PHA) stimulation occurred in the first 8 h after mitogen addition. Stimulation of lymphocytes by pokeweed mitogen, anti-human immunoglobulin, or bacterial lipopolysaccharide resulted in tRNA species which showed distinct differences from each other and also from the tRNAs produced by phytohemagglutinin stimulation. Leukemic lymphocyte tRNAs showed the most extensive differences in profile when compared with chromatograms from non-neoplastic cells stimulated by a variety of mitogens. Specific isoaccepting species of tyrosyl-, aspartyl-, and phenylalanyl-tRNAs were also compared in PHA-stimulated and resting lymphocytes and no differences were found. When these same species were studied in leukemic cells, tyrosyl-tRNA profiles were shifted to elute at a lower salt concentration, while the aspartyl-tRNA profile showed a new peak not present in noncancerous cells.     

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.     

Effects of lymphocyte activation on transfer RNAs

The influences of mitogen activation on the functional capacity of rat splenic tRNAs were evaluated. The specific amino acid acceptor activity, pmol of a specific amino acid accepted per nmol of tRNA, of isolated splenic tRNAs from in vivo Concanavalin A (37 h)-treated rats were up to 8 times the specific amino acid acceptor activities of splenic tRNAs from control rats. Control splenic tRNAs were treated with purified liver tRNA nucleotidyltransferase in vitro to repair the 3'[CCA] terminus of tRNAs, and subsequently assayed in an aminoacylation reaction. The specific amino acid acceptor activities were slightly increased over those tRNAs not repaired with tRNA nucleotidyltransferase, indicating the presence of a low level of defective but repairable tRNAs in the control rat spleen. Furthermore, our results indicate that cyclosporin A (inhibitor of lymphocyte activation) blocks the Concanavalin A stimulation of tRNA charging ranging from 16 to 93%.     

tRNA accumulation and suppression of the bldA phenotype during development in Streptomyces coelicolor

Streptomyces coelicolor undergoes distinct morphological changes as it grows on solid media where spores differentiate into vegetative and aerial mycelium that is followed by the production of spores. Deletion of bldA, encoding the rare tRNA(Leu) UAA, blocks development at the stage of vegetative mycelium formation. From previous data it appears that tRNA(Leu) UAA accumulates relatively late during growth while two other tRNAs do not. Here, we studied the expression of 17 different tRNAs including bldA tRNA, and the RNA subunit of the tRNA processing endoribonuclease RNase P. Our results showed that all selected tRNAs and RNase P RNA increased with time during development. However, accumulation of bldA tRNA and another rare tRNA(Leu) isoacceptor started at an earlier stage compared with the other tRNAs. We also introduced the bldA tRNA anticodon (UAA) into other tRNAs and introduced these into a bldA deletion strain. In particular, one such mutant tRNA derived from the tRNA(Leu) CAA isoacceptor suppressed the bldA phenotype. Thus, the bldA tRNA scaffold is not critical for function as a regulator of S. coelicolor cell differentiation. Further substitution experiments, in which the 5'- and 3'-flanking regions of the suppressor tRNA were changed, indicated that these regions were important for the suppression.     

Separation of isoacceptor cysteine transfer ribonucleic acids of bakers' yeasts

Summary Isoacceptor species of certain amino acidspecific transfer ribonucleic acids (tRNAs) were fractionated by gel permeation chromatography using Sephadex G-100. The separation is attributed to the 20% ethanol-1% NaCl solvent and to the characteristics of Sephadex. Isoacceptor tRNAs specific for cysteine, arginine, phenylalanine, and histidine were recovered from commercial tRNA of yeast by this method. Highly purified cysteine-specific tRNA, obtained by a method which would not be expected to separate isoacceptor molecules when fractionated by this procedure, was shown to contain two cysteine isoacceptor tRNAs.This investigation was supported in part by Public Health Service Research Grant AM-09131 from the National Institute of Arthritis and Metabolic Diseases.     

Novel anticodon composition of transfer RNAs in Micrococcus luteus, a bacterium with a high genomic G + C content. Correlation with codon usage.

The number and relative amount of isoacceptor tRNAs for each amino acid in Micrococcus luteus, a Gram-positive bacterium with high genomic G + C content, have been determined by sequencing their anticodon loop and its adjacent regions and by selective labelling of tRNAs. Thirty-one tRNA species with 29 different anticodon sequences have been detected. All the tRNAs have G or C at the anticodon first position except for tRNA(ICGArg) and tRNA(NGASer), in response to the abundant usage of NNC and NNG codons. No tRNA with the anticodon UNN capable of translating codon NNA has been detected, in accordance with a very low or zero usage of NNA codons. The relative amount of isoacceptor tRNAs for an amino acid determined by selective labelling strongly correlates with usage of the corresponding codons. On the basis of these and other observations in this and other eubacterial species, we conclude that the relative amount and anticodon composition of isoacceptor tRNA species are flexible, and their changes are mainly adaptive phenomena that have been primarily affected by codon usage, which in turn is affected by directional mutation pressure.     

Increased isoleucine acceptance by sulfur-deficient transfer RNA from Escherichia coli.

Sulfur-deficient tRNA, isolated from Escherichia coli HfrC, rel-, met-, cys-, lambda, after cysteine starvation, was found to have an increased acceptance of isoleucine in proportion to the deficiency of 4-thiouridine. Isoleucine acceptance was not altered in the presence of other amino acids of CTP, and the higher acceptance was observed over a wide range of magnesium, isoleucine, tRNA and enzyme concentrations. The Vmax value for sulfur-deficient tRNA was more than three times greater than observed for normal tRNA. Methylated albumin kieselguhr (MAK) chromatography revealed three isoacceptor peak for normal tRNA, while sulfur-deficient tRNA was missing tRNAile, and exhibited a larger, shifted peaks for tRNA normal tRNA, while sulfur-deficient tRNA was missing tRNAille 2, and exhibited a large shifted peak for tRNAile 3 . Treatment with crude RNA sulfurtransferase both lowered the isoleucine acceptance for sulfur-deficient tRNA to that seen for normal tRNA, and restored the missing isoacceptor on MAK. The possibility that thionucleotides may play a role in the aminoacylation of tRNAile in E. coli is discussed.     

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.     

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.     

Two kinetically distinct tRNAile isoacceptors in Escherichia coli C6.

The isoleucine acceptance of tRNA from Escherichia coli C6 was previously shown to be influenced by the synthetase level (Marashi, F. and Harris, C.L. 1977. Biochim. Biophys. Acta 477, 84-88). We show here that the increased acceptance observed at higher enzyme levels is accompanied by an increase in the aminoacylation of one tRNAile species. Hence, tRNAile, a minor species at low enzyme levels, is a major isoacceptor after full aminoacylation. The two major isoleucine species have been purified using a combination of BD-cellulose, DEAE-Sephadex A-50 and methylated albumin kieselguhr chromatography. tRNAile (1511 pmoles ile/A260 of tRNA) was found to be slowly acylated, with 2a Vmax one-seventh that observed with tRNAil3le (1475 pmoles ile/A260). Two-dimensional TLC analysis of RNase T2 digests revealed differences in nucleotide content between the purified tRNAs. These results are discussed in terms of the presence of slow and fast tRNAile species in E. coli.     

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.     

Superposition of two tRNA acceptor stem crystal structures: Comparison of structure, ligands and hydration

We solved the X-ray structures of two Escherichia coli tRNA^Ser acceptor stem microhelices. As both tRNAs are aminoacylated by the same seryl-tRNA-synthetase, we performed a comparative structure analysis of both duplexes to investigate the helical conformation, the hydration patterns and magnesium binding sites. It is well accepted, that the hydration of RNA plays an important role in RNA-protein interactions and that the extensive solvent content of the minor groove has a special function in RNA. The detailed comparison of both tRNA^Ser microhelices provides insights into the structural arrangement of the isoacceptor tRNA aminoacyl stems with respect to the surrounding water molecules and may eventually help us to understand their biological function at atomic resolution.     

Conservation of the relative tRNA composition in healthy and cancerous tissues

Elongation in protein translation is strongly dependent on the availability of mature transfer RNAs (tRNAs). The relative concentrations of the tRNA isoacceptors determine the translation efficiency in unicellular organisms. However, the degree of correspondence of codons and the relevant tRNA isoacceptors serves as an estimator for translation efficiency in all organisms. In this study, we focus on the translational capacity of the human proteome. We show that the correspondence between the codon usage and tRNAs can be improved by combining experimental measurements with the genomic copy number of isoacceptor groups. We show that there are technologies of tRNA measurements that are useful for our analysis. However, fragments of tRNAs do not agree with translational capacity. It was shown that there is a significant increase in the absolute levels of tRNA genes in cancerous cells in comparison to healthy cells. However, we find that the relative composition of tRNA isoacceptors in healthy, cancerous, or transformed cells remains almost identical. This result may indicate that maintaining the relative tRNA composition in cancerous cells is advantageous via its stabilizing of the effectiveness of translation.     

The specificity of aminoacylation: a tRNA-tRNA interaction model.

Most of the isoacceptor species for a particular tRNA can be classified according to the middle base in the anticodon together with the fourth base in the amino acid stem. These specifying nucleotides would operate if a tRNA-tRNA interaction occurs on the aminoacyl-tRNA synthetase so that the anticodon of one tRNA molecule faces the fourth base of the other tRNA molecule. This model explains most of the misacylation reactions or changes in aminoacylation after mutation or chemical modifications of tRNAs. It also provides an explanation for biochemical properties of the aminoacyl-tRNA synthetases such as the presence of two active sites, and for the high fidelity of the aminoacylation. It may give insight into the origin and stability of the genetic code.