tRNA Population Research Articles

Expression of the Synechocystis sp. strain PCC 6803 tRNA(Glu) gene provides tRNA for protein and chlorophyll biosynthesis

In the cyanobacterium Synechocystis sp. strain PCC 6803 (Synechocystis 6803) delta-aminolevulinic acid (ALA), the sole precursor for the synthesis of the porphyrin rings of heme and chlorophyll, is formed from glutamate activated by acylation to tRNA(Glu) (G. P. O'Neill, D. M. Peterson, A. Schön, M. W. Chen, and D. Söll, J. Bacteriol. 170:3810-3816, 1988; S. Rieble and S. I. Beale, J. Biol. Chem. 263:8864-8871, 1988). We report here that Synechocystis 6803 possesses a single tRNA(Glu) gene which was transcribed as monomeric precursor tRNA and matured into the two tRNA(Glu) species. They differed in the extent of modification of the first anticodon base, 5-methylaminomethyl-2-thiouridine (O'Neill et al., 1988). The two tRNA species had equivalent capacities to stimulate the tRNA-dependent formation of ALA in Synechocystis 6803 and to provide glutamate for protein biosynthesis in an Escherichia coli-derived translation system. These results are in support of a dual role of tRNA(Glu). The levels of tRNA(Glu) were examined by Northern (RNA) blot analysis of cellular RNA and by aminoacylation assays in cultures of Synechocystis 6803 in which the amount of chlorophyll synthesized was modulated over a 10-fold range by various illumination regimens or by the addition of inhibitors of chlorophyll and ALA biosynthesis. In these cultures, the level of tRNA(Glu) was always a constant fraction of the total tRNA population, suggesting that tRNA(Glu) and chlorophyll levels are regulated independently. In addition, the tRNA(Glu) was always fully aminoacylated in vivo.

Transfer RNAs of potato (Solanum tuberosum) mitochondria have different genetic origins

Total transfer RNAs were extracted from highly purified potato mitochondria. From quantitative measurements, the in vivo tRNA concentration in mitochondria was estimated to be in the range of 60 microM. Total potato mitochondrial tRNAs were fractionated by two-dimensional polyacrylamide gel electrophoresis. Thirty one individual tRNAs, which could read all sense codons, were identified by aminoacylation, sequencing or hybridization to specific oligonucleotides. The tRNA population that we have characterized comprises 15 typically mitochondrial, 5 'chloroplast-like' and 11 nuclear-encoded species. One tRNA(Ala), 2 tRNAs(Arg), 1 tRNA(Ile), 5 tRNAs(Leu) and 2 tRNAs(Thr) were shown to be coded for by nuclear DNA. A second, mitochondrial-encoded, tRNA(Ile) was also found. Five 'chloroplast-like' tRNAs, tRNA(Trp), tRNA(Asn), tRNA(His), tRNA(Ser)(GGA) and tRNA(Met)m, presumably transcribed from promiscuous chloroplast DNA sequences inserted in the mitochondrial genome, were identified, but, in contrast to wheat (1), potato mitochondria do not seem to contain 'chloroplast-like' tRNA(Cys) and tRNA(Phe). The two identified tRNAs(Val), as well as the tRNA(Gly), were found to be coded for by the mitochondrial genome, which again contrasts with the situation in wheat, where the mitochondrial genome apparently contains no tRNA(Val) or tRNA(Gly) gene (2).

Translational readthrough of the murine leukemia virus gag gene amber codon does not require virus-induced alteration of tRNA.

An in vitro system to assay translational readthrough of the UAG termination codon at the murine leukemia virus (MuLV) gag-pol junction was developed by using rabbit reticulocyte lysates programmed by SP6-generated Moloney MuLV gag-pol mRNA. Under conditions in which the suppressor activity of the lysate was dependent on addition of tRNA, it could be shown that readthrough synthesis was stimulated to approximately the same extent by equivalent amounts of tRNA from MuLV-infected and uninfected NIH 3T3 cells. Analysis of glutamine tRNA, which mediates suppression in vivo, showed that the level of glutamine acceptor activity and the chromatographic profile of glutamine isoacceptors were unchanged following virus infection. On the basis of these results, we conclude that the suppressor tRNA occurs normally within the tRNA population of uninfected cells and need not be induced in response to virus infection.

Specifie codon usage pattern and its implications on the secondary structure of silk fibroin mRNA

We have identified two distinctive regions of the repetitive unit nucleotide sequence of fibroin mRNA of Bombyx mori. The codon usage for the major amino acids, glycine, alanine and serine is distinctly different in these two regions, indicating that it is determined by the fibroin mRNA or gene structure but not by the tRNA population. Comparative computer analyses of nucleotide substitutions in the unit sequence suggest that selection has operated on the codon usage to optimize the secondary structure characteristic of the fibroin mRNA.

Relative content of isoaccepting tRNAs for glycine and proline in avian tendon cells with different rates of procollagen synthesis

The relative amounts of iso-tRNAs Gly and iso-tRNAs Pro existing in chick embryo tendon are indicative of a specialization of the tRNA population for collagen synthesis. These amounts are not modified (i) in primary avian tendon (PAT) cells in culture for which the procollagen production varies from about 10% of total protein synthesis to 60% and (ii) in tendons from immature chicks, which show a 3-fold decrease of procollagen production with increasing age. The characteristic tRNA pattern was not maintained in cells which had lost the ability to make high levels of collagen as observed in the cases of: (i) PAT cells reaching confluency; (ii) virus-transformed PAT cells and (iii) tendon from adult chick. Our data are consistent with the idea that tendon tRNA specialization for collagen synthesis is a differentiation feature independent of the expression level of the collagenic function but related to its maintenance.

The independent regulation of and tRNAAsn synthesis during Friend cell erythroid differentiation

In this report, we have compared the changes in the production of tRNA(iMet) (initiator tRNA(Met] and tRNA(Asn), which occur during erythroid differentiation in the Friend erythroleukemia cell. The relative steady-state concentration of these two tRNAs (relative to the total tRNA population) was measured by aminoacylation. The results show that while the relative steady-state concentration of tRNA(iMet) changes very little in the cytoplasmic tRNA population, the relative concentration of tRNA(Asn) decreases during the first two days of differentiation and then undergoes an increase. This difference in the behavior of these two tRNAs is also seen when their relative concentrations in newly synthesized tRNA is examined. When tRNA is labeled with tritiated uridine for 24 h in vivo prior to isolation, the hybridization of this labeled tRNA to filter-bound tRNA genes shows that the relative concentration of tRNA(iMet) in newly synthesized tRNA changes very little, while the relative concentration of newly synthesized tRNA(Asn) again decreases through the first 2 days of differentiation, and then undergoes a smaller increase. Thus, the production of these two tRNAs appears to be independently regulated. Independent regulation of synthesis is also observed when examining the production of these two tRNAs in isolated nuclei. During erythroid differentiation, the relative synthesis of tRNA(iMet) (relative to total nuclear RNA synthesis) remains constant, while the relative synthesis of tRNA(Asn) undergoes periodic increases and decreases in value.

Distribution of isoaccepting tRNAs and codons for proline and glycine in collagenous and noncollagenous chicken tissues

The relation between codon usage and tRNA content for proline and glycine, the major constituents of collagen, was studied in two tissues: the magnum of laying hen oviduct and the leg tendons of chick embryo where collagen is produced. Although the relative contents of tRNA GCC Gly and tRNA IGG Pro in tendons, as compared to magnum indicate a specialization of the tRNA population for collagen synthesis, the distribution of the preponderant codons in collagen mRNA is correlated but at a lesser extent to that of their cognate tRNAs.

TRNA nucleotidyltransferase is not essential for Escherichia coli viability.

The role of tRNA nucleotidyltransferase in Escherichia coli has been uncertain because all tRNA genes studied in this organism already encode the -C-C-A sequence. Examination of a cca mutant, originally thought to contain 1-2% enzyme activity, indicated that it actually produces an inactive fragment of 40 kd compared to 47 kd for the wild-type enzyme due to a nonsense mutation in its cca gene. To confirm that the residual activity in extracts of this strain is due to another enzyme, and that tRNA nucleotidyltransferase is non-essential, we have interrupted the cca gene in vitro, and transferred this mutant gene to a variety of strains. In all cases mutant strains are viable, although as much as 15% of the tRNA population contains defective 3' termini, and no tRNA nucleotidyltransferase is detectable. Mutant strains grow slowly, but can be restored to more normal growth by a relA mutation or by a decrease in RNase T activity. In the latter case the amount of defective tRNA decreases dramatically. These findings indicate that tRNA nucleotidyltransferase is not essential for E. coli viability, and therefore, that all essential tRNA genes in this organism encode the -C-C-A sequence.

Differences in the tRNA population between summer and winter acclimatized carp

1. 1. Transfer RNA isolated from the livers of winter and summer acclimatized male carps was fractionated by two-dimensional polyacrylamide electrophoresis. 2. 2. Significant changes in the distribution of the tRNA isoacceptor species were observed. 3. 3. The adjustment of the tRNA population to the needs of the protein synthetic machinery during the acclimatization process correlates with the cyclic carp liver cell ultrastructural changes previously observed, and provides a new approach to gain knowledge about the mechanisms involved in the compensatory response to thermal adaptation.

Adjustment of the tRNA population to the codon usage in chloroplasts.

In chloroplasts there is a correlation between the amounts of tRNAs specific for a given amino acid and the codons specifying this amino acid. Furthermore, for the amino acids coded for by more than one codon, the population of isoaccepting tRNAs is adjusted to the frequency of synonymous codons used in chloroplast protein genes. A comparison by two-dimensional gel electrophoresis of the tRNA populations extracted from chloroplasts and from chloroplast polysomes shows that all chloroplast tRNAs are involved in protein biosynthesis.

The measurement of the production of tRNA Met1 in the Friend erythroleukemia cell

We have used the gene for tRNA Met 1 as a hybridization probe to measure the production of tRNA Met 1 in the Friend erythroleukemia cell. In this cell, the relative concentration of tRNA Met 1 (i.e., the percentage of total steady-state tRNA representing tRNA Met 1) is 1.60 ± 0.18. To study the relative synthesis of tRNA Met 1, cells were labeled in vivo with [ 3H]uridine for periods ranging from 4 to 24 h, and the tRNA was isolated. The fraction of newly-synthesized tRNA representing tRNA Met 1 (1.72% ± 0.11) does not change when different in vivo labeling times are used. This value is similar to the relative concentration of tRNA Met 1 in the older steady-state tRNA (1.61% ± 0.18). The similar relative synthesis values using different labeling times, plus evidence presented that the total tRNA population decays homogeneously ( t 1 2 = 110 h ) indicate that tRNA Met 1 has a cytoplasmic stability similar to the general tRNA population, and that its concentration relative to the tRNA population is established within the nucleus or soon after exiting the nucleus. Measurements of the synthesis of tRNA Met 1 in isolated nuclei, relative to the synthesis of total RNA polymerase III transcripts, showed that this relative synthesis (0.291% ± 0.017) is only 17% of the relative concentration of tRNA Met 1 in the cytoplasm, which may reflect the presence of sequences other than tRNA in total nuclear polymerase III transcripts.

Transfer ribonucleic acid populations in concanavalin-A-stimulated bovine lymphocytes

Transfer RNA isolated from lymphocytes stimulated by concanavalin A and that from resting cells were compared with respect to amino-acid acceptance, integrity of the CCA-terminus, extent of modification and isoacceptor distribution. Following growth stimulation the overall amino-acid acceptance of the tRNA is elevated, in particular the relative acceptor activities for threonine and arginine are increased. The reduced acceptor activity of the tRNA from the quiescent cells is not due to a preferential degradation of the CCA-end, since it persits even in the presence of ATP(CTP):tRNA nucleotidyltransferase. We therefore conclude that this reduced activity is caused by structural differences of the tRNAs. The content of modified nucleotides in newly synthesized tRNA from lymphocytes cultured in the presence and absence of concanavalin A was determined. tRNA from resting cells was found to be undermodified with respect to pseudouridine and dihydrouridine. Upon monitoring the tRNA isoacceptor distribution by affinity chromatography on immobilized elongation factor Tu and subsequent two-dimensional gel electrophoresis, a preferential synthesis of particular lysine- and threonine-accepting tRNAs was observed upon mitogenic stimulation. Evidently, a specific tRNA population is needed by the proliferating cells. These results are discussed in view of the hypothesis that the commitment of lymphocytes to proliferation is at least in part under translational control.

Purification and characterization of Escherichia coli RNase T.

RNase T, a nuclease thought to be involved in end-turnover of tRNA, has been purified about 4,000-fold from extracts of Escherichia coli. At this stage of purification, the enzyme was judged to be at least 95% pure based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight of RNase T determined from gel filtration and sedimentation analyses is about 50,000, whereas the monomer molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 25,000, suggesting that the protein is an alpha 2 dimer. Purified RNase T is extremely sensitive to inactivation by oxidation, sulfhydryl group reagents, and temperature. The ribonuclease activity against tRNA-C-C-[14C]A is optimal at pH 8-9 in the presence of 2-5 mM MgCl2 and ionic strengths of less than 50mM. Although RNase T is highly specific for intact tRNA-C-C-A as a substrate and can hydrolyze all species in a mixed population of tRNA, it is inhibited by other RNAs, such as poly(A), rRNA, 5 S RNA, and tRNA-C-C. RNase T is an exoribonuclease which initiates attack at a free 3' terminus of tRNA and releases AMP; aminoacyl-tRNA is not a substrate. The role of RNase T in the end-turnover of tRNA and its possible involvement in other aspects of RNA metabolism are discussed.

Prolactin homogeneously induces the tRNA population of mouse mammary explants

Explants of mouse mammary glands were cultured with and without prolactin in the presence of inorganic [ 32P] to estimate the effect of prolactin on tRNA synthesis. Labeled tRNA was extracted and characterized by two-dimensional gel electrophoresis. tRNA synthesis was 2–3 fold greater in the presence of prolactin; and the synthesis rate of each resolvable tRNA species was increased proportionally. tRNA populations from mouse mammary tissues at three stages of development were also examined. Alterations were noted between early pregnant and fully lactating tissues. The results of this study provide evidence that the tRNA population, which is known to be “specialized” for casein synthesis in the mammary gland, is determined as the gland develops and prepares for lactation.

Analysis of the codon bias in E. coli sequences.

Fifty-three gene sequences from E. coli containing 18,288 reading frame triplets have been characterized according to the nature and level of average codon preference. The distribution of average preferences is bimodal, with approximately half the genes using an average of only 36 codons, and the remainder just 42 codons. There is a high correlation between the level of codon bias, the tRNA population and the abundance of protein product, indicating biased patterns are exploited by the cell for the production of widely different levels of gene product. This relationship is especially striking in genes involved in the production of components for transcription and translation. Overall, the genes for these processes generate some five-fold more protein than the average in the genome, and use about five fewer codons. The very high codon bias found in the RNA polymerase gene thus provides a simple, autogenous mechanism for the coordinate synthesis of these components and RNA polymerase. A surprisingly high level of codon probability is also found in triplets of the complement of coding sequences. This is apparently due to the evolutionary dispersion of coding sequences and/or the requirement for increased levels of secondary structure in messenger RNAs.

Identification and synthesis of a naturally occurring selenonucleoside in bacterial tRNAs: 5-[(methylamino)methyl]-2-selenouridine.

Escherichia coli, Clostridium sticklandii, and Methanococcus vannielii synthesize 75Se-labeled amino acid transfer ribonucleic acids [( 75Se]tRNAs) when grown with low levels (approximately equal to 1 microM) of 75SeO32-. When E. coli [75Se]tRNA was digested to nucleosides and analyzed by reversed-phase high-performance liquid chromatography, a single selenonucleoside accounted for 70-90% of the 75Se label in the bulk tRNA. This nucleoside was shown to be indistinguishable in a number of its properties from authentic 5-[(methylamino)methyl]-2-selenouridine. Preparation of the authentic selenonucleoside was accomplished and the synthetic compound characterized by its UV and 1H NMR spectral properties. The new selenonucleoside also accounted for 40-60% of the 75Se found in [75Se]tRNA from C. sticklandii or M. vannielii. Each of these anaerobic bacteria contains one additional selenonucleoside in their tRNA populations distinct from 5-[(methylamino)methyl]-2-selenouridine. Pure seleno-tRNAGlu isolated from C. sticklandii contains one 5-[(methylamino)methyl]-2-selenouridine and one 4-thiouridine per tRNA molecule.

Distribution of two selenonucleosides among the selenium-containing tRNAs from Methanococcus vannielii

Naturally occurring seleno-tRNAs from Methanococcus vannielii account for 13-20% of the total tRNA population. Two different selenium-modified nucleosides were detected in these seleno-tRNAs. Of the total selenium incorporated, about 60% is present in 5-methylaminomethyl-2-selenouridine, and the other 40% occurs in a second selenonucleoside with spectral characteristics typical of a derivative of 2-selenouridine. The 5-methylaminomethyl-2-selenouridine was found in the seleno-tRNA species present in the early fractions of a linear salt gradient elution profile from a reversed-phase chromatographic system 5 (RPC-5) column, whereas the second selenonucleoside occurred in the tRNA species eluted late in the profile.

Function and fidelity of aging tRNA: In vivo acylation, analog discrimination, synthetase binding, and in vitro translation

The liver transfer RNAs for valine and lysine were completely acylated in vivo, as judged by periodate oxidation, at 4 and 24 months of age in male Sprague-Dawley rats. In vitro acylation capacity for whole tRNA populations from rat livers is decreased, but this is evidently not deleterious in vivo. Several halogenated phenylalanines were synthesized and their effects upon acylation capacity for phenylalanine were examined. Synthetases bound to young (3 month) and old (24 month) tRNAs discriminated differently between p-chlorophenylalanine and authentic phenylalanine; synthetase with young tRNA was less able to discriminate than with old tRNA. Purified tRNA phe from old rats did not form ultraviolet-induced crosslinks to purified phenylalanyl tRNA synthetase as well as young tRNA phe. In vitro translation of encephalomyocarditis virus, hemoglobin, and ovalbumin mRNAs was effective, using tRNAs of young or old Sprague-Dawley or Fischer 344 rat livers, although, when the old tRNA was supplied, the production synthesized per unit tRNA was reduced. All of the protein products were synthesized with all tRNAs, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We conclude that tRNA is capable of normal functions in livers of aging rats, is probably modification deficient, and is unlikely to produce protein errors.

The isolation and measurement of tRNAmeti using RNA/DNA hybridization.

A pBR322 plasmid containing the initiator tRNAmet gene of Xenopus (pt145 - donated by Stuart Clarkson) will specifically bind to mouse initiator tRNAmet (tRNAmeti) when total mouse tRNA, extracted from uninduced Friend erythroleukemia cells, is hybridized to the gene probe. One dimensional electrophoresis of the hybridizing tRNA in 20% polyacrylamide reveals one major band (95%) and a minor band. The hybridizing tRNA has been identified as initiator tRNAmet by RNA sequencing. Hybridization of tRNAtotal to another plasmid containing the Xenopus gene for tRNAasn results in two bound species with different electrophoretic mobilities than the tRNA bound to the initiator tRNAmet gene. pt145 has been used to measure the steady state concentration of initiator tRNAmet in the uninduced and erythroid Friend cell, and in the unfertilized egg and 21 h blastula of the sea urchin. Initiator tRNAmet represents 0.91% and 0.52% of the tRNA populations extracted from uninduced and erythroid Friend cells, respectively. Based upon the total tRNA content per cell, there is a 3.8 fold decrease in initiator tRNAmet per cell during erythroid differentiation. tRNA extracted from unfertilized eggs and 21 h blastula of the sea urchin both have 0.5% of total tRNA as initiator tRNAmet (approximately 1.5 pg).

Perturbation of the mitochondrial lysine tRNA population by virus-induced transformation or stress of mammalian cells: functional properties and nucleotide sequence of a mitochondrially associated lysine tRNA.

Eleven isoaccepting lysine tRNAs from mammalian sources are demonstrable by RPC-5 chromatography and polyacrylamide gel electrophoresis. The appearance and amounts of these isoacceptors varies with the source and growth state of cells. One isoacceptor, tRNALys6, observed in preparations of tRNA from some virus-transformed cells in culture, has been characterized by determining functional properties, cellular location, and its nucleotide sequence. tRNALys6 responds primarily to the lysine codon AAA, but it is not used efficiently in a wheat germ translational system in vitro. Compared with lysine isoacceptors 1, 2, 4, 5a, and 5, [3H]lysine appears in vivo in tRNALys6 with a delay of about 3 h. This delay may in part be a result of a less functional tRNA, but a compartmented state of tRNALys6 also appears to be important. tRNALys6 is associated with mitoplasts prepared from KA31 fibroblasts. The nucleotide sequence of tRNALys6 was determined by rapid postlabeling procedures involving limited hydrolysis in formamide, 32P-labeling of 5' ends of fragments with polynucleotide kinase, separation of the nested set of fragments in polyacrylamide denaturing gels, release of 5'-labeled nucleotides with RNase T2, and identification of the released nucleotides by chromatography on PEI cellulose. Confirmation of the positions of major nucleotides was done by using limited digestions by RNases of tRNALys6 labeled with 32P on the 3' terminus in a gel readout procedure. The nucleotide sequence of tRNALys6 differs from that of cytoplasmic lysine tRNAs and mammalian mitochondrial lysine tRNAs. It contains U*, an unidentified modified uridine occurring in the anticodon of some mitochondrial tRNAs. tRNALys6 appears to occur in very limited amounts, or not at all, in most cells unless stressed, but when present it is associated with mitochondria, although it is probably coded in the nucleus.