tRNA Fraction Research Articles

16] Isolation of mitochondrial tRNAs from human cells

This chapter explains the isolation of mitochondrial tRNAs from human cells. Mammalian mitochondrial tRNAs possess many unique sequence, structural, and functional properties. All mammalian mitochondrial DNAs (mtDNAs) code for 22 tRNAs, which are sufficient for the translation of the mtDNA-encoded mRNAs, because a single tRNA is able to read all codons of a four-codon family. In contrast to prokaryotic, eukaryotic nuclear, and chloroplast tRNAs, whose structures are remarkably conserved, there are numerous sequence and structural alterations found among mammalian mitochondrial tRNAs. These include changes in highly conserved nucleotides or nucleotide base modifications, variations in the sizes of stem and loop structures, and alterations in conserved tertiary interactions. The chapter describes procedures for the isolation of single tRNA species based on a variety of fractionation methods. tRNA fraction, isolated by diethylaminoethyl (DEAE) cellulose, is subsequently further fractionated by electrophoresis through successive polyacrylamide gels. In addition, the chapter also describes procedure for cultured mammalian cells that allows the isolation of a highly purified total mitochondrial tRNA preparation. This procedure has been used previously to investigate the steady-state levels and metabolic properties of tRNAs isolated from HeLa cell mitochondria.

Differential up-regulation by tRNAs of ribosome-inactivating proteins

Some plant ribosome-inactivating proteins (RIPs) with RNA- N-glycosidase activity on 28S RNA require, for the inactivation of ribosomes, the presence of macromolecular cofactors present in post-ribosomal supernatants. In the case of gelonin one of the cofactors is tRNA Trp lacking one or two nucleotides at the 3′-CCA end [Brigotti, M., Carnicelli, D., Alvergna, P., Pallanca, A., Lorenzetti, R., Denaro, M., Sperti, S. and Montanaro, L. (1995) Biochem. J. 310, 249–253]. In the present study it is shown that tRNAs are involved in the up-regulation of all the cofactor-requiring RIPs up to now identified (agrostin, barley RIP, PAP and tritin, besides gelonin). Polyacrylamide gel electrophoresis shows that tRNA fractions with different mobilities stimulate different RIPs. With the identification of agrostin, the cofactor-requiring RIPs (italics) add to five out of a total of thirteen investigated: barley RIP, bryodin-R, gelonin, lychnin, momordin, momorcochin-S, PAP, saporin-6, tritin [Carnicelli, D., Brigotti, M., Montanaro, L. and Sperti, S. (1992) Biochem. Biophys. Res. Commun. 182, 579–582], agrostin, luffin, trichokirin and trichosanthin (present study).

Separation of Rat Liver Cytoplasmic Trnas by High Performance Liquid Chromatography

Abstract Experiments aimed at developing a chromatographic map of cytoplasmic tRNA population from rat liver are described. HPLC tRNA fractionation by a linear gradient of acetonitrile/ammonium acetate, results in a chromatogram with more than 60 significant peaks, having area peak percentages ranging from 0.001 to 5.0. This paper presents evidence that each peak corresponds to a specific tRNA species. Specificity of tRNA-peak assignment was established by charging total tRNA with a mixture of 19 unlabeled aminoacids plus a radioactive aminoacid, and isolating the radioactive aminoacylated iso-tRNAs by gel electrophoresis, followed by HPLC acetonitrile gradient. The radioactive aminoacyl-tRNA species were eluted as well-behaved peaks. Here we describe the analysis of tRNATrp, which gave 2 well separated radioactive peaks, most likely corresponding to tRNATrp isoacceptor species postulated by the wobble hypothesis. Taken together, the present data validate this procedure for monitoring quantitatively and q...

A deviation from the universal genetic code in Candida maltosa and consequences for heterologous expression of cytochromes P450 52A4 and 52A5 in Saccharomyces cerevisiae.

We demonstrate that serine instead of leucine is specified by the CUG codon in the yeast Candida maltosa. Evidence for this deviation from the universal genetic code was obtained by means of in vitro translation experiments. Depending on the cell-free system used, either serine, in the C. maltosa system, or leucine, in the control with the conventional wheat germ system, was found to be incorporated into the translation products of artificial CUG-containing mRNAs. Moreover, we were able to transfer the non-universal decoding of CUG to the wheat germ system by adding a tRNA fraction isolated from C. maltosa. This finding indicates the presence in C. maltosa of an unusual serine tRNA that recognizes CUG. As a consequence of the altered genetic code, expression in Saccharomyces cerevisiae of C. maltosa cytochrome P450 genes required an exchange of their CTG triplets by TCT encoding serine in order to produce the authentic proteins. In contrast, heterologous expression of the original C. maltosa genes resulted in the formation of still active but unstable enzymes probably subject to selective proteolysis in the host cells.

Fractionation of rat liver tRNA by reversed-phase high performance liquid chromatography: isolation of Iso-tRNAs(Pro).

This paper illustrates the fractionation of cytoplasmic transfer ribonucleic acid from rat liver by reversed-phase high performance liquid chromatography using a gradient of acetonitrile/ammonium acetate. The procedure is fast, highly reproducible, and gives an excellent resolution of the numerous tRNA population: about 50 peaks with area peak percentages ranging from 0.001 to 5 can be monitored. Uncharged tRNA preparations exhibited a chromatographic profile different from aminoacylated tRNA, thus suggesting a possible strategy to distinguish between aminoacylated and nonacylated tRNA species. Moreover, a first approach to map the HPLC peaks was attempted by chromatographing preparations of tRNA which had been aminoacylated with individual 3H-labeled aminoacids. Here is reported the case of tRNA(Pro), which gave three well separated radioactive peaks, most likely corresponding to tRNA(Pro) isoacceptor species.

Detection of Human tRNAs with Antisense Oligonucleotides

Regulated expression and modification of tRNA isoacceptors may play an important role in the control of gene expression during such processes as differentiation and immune activation. However, the development of techniques for the identification and quantitation of multiple tRNA isoaccepting species has been hindered by the relative physicochemical similarity among individual isoacceptors and their high degree of post-transcriptional modification. We have used antisense DNA oligonucleotides derived from the T stem to acceptor stem region of six human tRNAs and one murine tRNA to detect individual tRNA isoacceptors in slot blots, Northern blots, and dot blots of human tRNA. This hybridization protocol was used in combination with tRNA fractionation by electrophoresis on a partially denaturing gel by reversed-phase low pressure chromatography and reversed-phase HPLC to identify multiple tRNA isoacceptors in a single sample of tRNA. Using this technique, it should be possible to monitor changes in the cellular tRNA repertoire that may be involved in the regulation of gene expression.

TRNA fractionation by HPLC.

tRNA fractionation by HPLC.

An enzymatic method for the estimation of L-leucine in rat blood

A specific enzymatic method for the routine measurement of L-leucine in blood samples is presented. The method uses a commercial preparation of tRNAs and amino acyl-tRNA synthetases for the specific loading of L-leucine into the tRNALeu present, competing with carrier-free L-[U-14C]leucine. The radioimmunoassay-like plot of radioactivity found in the acid-insoluble (tRNA) fraction was used to determine the amount of unlabelled L-leucine of the samples when compared against a standard curve. The interference of L-isoleucine, L-valine and L-alanine was very low.

Characterization of the process of 5-aminolevulinic acid formation from glutamate via the C 5 pathway in Clostridium thermoaceticum

1. 1. In vitro formation of 5-aminolevulinic acid (ALA) from glutamate required two enzyme fractions, separable on Blue Sepharose affinity chromatography, and a tRNA fraction, which can be replaced by Escherichia coli tRNA Glu in the reconstituted assay. 2. 2. Gabaculine was shown to inhibit ALA formation in the complete assay as well as in a defined system consisting of only glutamate-1-semialdehyde and the enzyme fraction not retained on Blue Sepharose. 3. 3. The results indicate that the enzyme system supporting ALA formation in Clostridium thermoaceticum is very similar to the tRNA Glu-dependent C 5 pathway in plant plastids.

Growth rate dependence of transfer RNA abundance in Escherichia coli.

We have tested the predictions of a model that accounts for the codon preferences of bacteria in terms of a growth maximization strategy. According to this model the tRNA species cognate to minor and major codons should be regulated differently under different growth conditions: the isoacceptors cognate to major codons should increase at fast growth rates while those cognate to minor codons should decrease at fast growth rates. We have used a quantitative Northern blotting technique to measure the abundance of the methionine and the leucine isoacceptor families over growth rates ranging from 0.5 to 2.1 doublings per hour. Five tRNA species that are cognate to major codons (tRNA(eMet), tRNA(1fMet), tRNA(2fMet), tRNA(1Leu) and tRNA(3Leu) increase both as a relative fraction of total tRNA and in absolute concentration with increasing growth rates. Three tRNA species that are cognate to minor codons (tRNA(2Leu), tRNA(4Leu) and tRNA(5Leu) decrease as a relative fraction of total RNA and in absolute concentration with increasing growth rates. These data suggest that the abundances of groups of tRNA species are regulated in different ways, and that they are not regulated simply according to isoacceptor specificity. In particular, the data support the growth optimization model for codon bias.

Relationship between protein synthesis and concentrations of charged and uncharged tRNATrp in Escherichia coli.

We have continuously monitored Trp-tRNA(Trp) concentrations in vivo and, in the same cultures, measured rates of protein synthesis in isogenic stringent and relaxed strains. We have also manipulated cellular charged and uncharged [tRNA(Trp)] by two means: (i) the strain used contains a Trp-tRNA synthetase mutation that increases the Km for Trp; thus, varying exogenous Trp varies cellular Trp-tRNA(Trp); and (ii) we have introduced into the mutant strain a plasmid containing the tRNA(Trp) gene behind an inducible promoter; thus, total [tRNA(Trp)] also can be varied depending on length of induction. The use of these conditions, combined with a previously characterized assay system, has allowed us to demonstrate that (i) the rate of incorporation of Trp into protein is proportional to the fraction of tRNA(Trp) that is charged; for any given total [tRNA(Trp)], this rate is also proportional to the [Trp-tRNA(Trp)]; (ii) uncharged tRNA(Trp) inhibits incorporation of Trp into protein; and (iii) rates of incorporation into protein of at least two other amino acids, Lys and Cys, are also sensitive to [Trp-tRNA(Trp)] and are inhibited by uncharged tRNA(Trp). Our results are consistent with models of translational control that postulate modulating polypeptide chain elongation efficiency by varying concentrations of specific tRNAs.

Chromatography ofE. colitRNA on 5–20 μm Agarose Beads

Abstract Escherichia coli transfer RNAs have been separated by chromatography on 5–20 μm beads of divinylsulfone-crosslinked agarose with the use of isocratic elution combined with a negative salt gradient. For example, tRNALeu was resolved into six isoacceptor species and tRNASer into four. Leucine-charged tRNA's were eluted after their corresponding uncharged isoacceptor species. By optimizing flow rate, column length and elution buffer the fractionation of tRNA could be performed within 3 hours.

Location of a single tRNA-His gene on the master chromosome of sunflower mitochondrial DNA

A highly purified tRNA fraction isolated from sunflower mitochondria was used to probe a restriction fragment library of sunflower mitochondrial DNA. Approximately 20 different clones were detected. The 0.2-kbp HindIII insert of recombinant plasmid pHD500 was sequenced revealing the presence of a tRNA-His gene with 97% similarity to the equivalent of maize mitochondria and chloroplast. The single copy gene appears to be located at least 30 kbp distant from regions of high homology with cpDNA in the mitochondrial genome. This observation and other evidence (analysis of flanking regions and hybridization to cpDNA) suggest that the gene probably does not belong to a chloroplast insertion. The possible expression of the tRNA-His gene is discussed.

Micropreparative separation of transfer ribonucleic acids by high-performance liquid chromatography

A method was developed for the micropreparative separation of individual species of tRNA using reversed-phase high-performance liquid chromatography on large pore spherical silica bonded with C 3 alkyl chains. Columns were eluted with linear gradients of decreasing sodium chloride and increasing methanol concentrations. The decreasing salt gradient gradually abolished hydrophobic interactions and a significantly higher selectivity was thus obtained when compared with increasing gradients of salts usually employed in reversed-phase separations of tRNA. The acceptance of tRNA fractions was tested by charging them with fifteen different amino acids. Significantly different separations were obtained with tRNA from Escherichia coli and from rat liver. tRNA Glu and tRNA Tyr from E. coli were obtained in a pure form, all other tRNAs were more or less contaminated by adjoining tRNAs for other amino acids. Rechromatography under suitable isocratic conditions was required to obtain pure tRNA species from rat liver. Isoaccepting tRNAs for several amino acids were separated from rat liver. The method described seems suitable for preliminary fractionations of complex mixtures of tRNA and for a simple purification of isoaccepting species if the presence of tRNAs for other amino acids is not an hindrance.

Characterization of the RNA Required for Biosynthesis of δ-Aminolevulinic Acid from Glutamate

The heme and chlorophyll precursor delta-aminolevulinic acid acid (ALA) is formed in plants and algae from glutamate in a process that requires at least three enzyme components plus a low molecular weight RNA which co-purifies with the tRNA fraction during DEAE-cellulose column chromatography. RNA that is effective in the in vitro ALA biosynthetic system was extracted from several plant and algal species that form ALA via this route. In all cases, the effective RNA contained the UUC glutamate anticodon, as determined by its specific retention on an affinity resin containing an affine ligand directed against this anticodon. Construction of the affinity resin was based on the fact that the UUC glutamate anticodon is complementary to the GAA phenylalanine anticodon. By covalently linking the 3' terminus of yeast tRNA(Phe(GAA)) to hydrazine-activated polyacrylamide gel beads, a resin carrying an affine ligand specific for the anticodon of tRNA(Glu(UUC)) was obtained. Column chromatography of plant and algal RNA extracts over this resin yielded a fraction that was highly enriched in the ability to stimulate ALA formation from glutamate when added to enzyme extracts of the unicellular green alga Chlorella vulgaris. Enhancement of ALA formation per A(260) unit added was as much as 50 times greater with the affinity-purified RNA than with the RNA before affinity purification. The affinity column selectively retained RNA which supported ALA formation upon chromatography of RNA extracts from species of the diverse algal groups Chlorophyta (Chlorella Vulgaris), Euglenophyta (Euglena gracilis), Rhodophyta (Cyanidium caldarium), and Cyanophyta (Synechocystis sp. PCC 6803), and a higher plant (spinach). Other glutamate-accepting tRNAs that were not retained by the affinity column were ineffective in supporting ALA formation. These results indicate that possession of the UUC glutamate anticodon is a general requirement for RNA to participate in the conversion of glutamate to ALA in plants and algae.

Biochemical research on oogenesis: distribution of tRNA-linked peptides and proteins in previtellogenic oocytes of Xenopus laevis

Peptides and proteins were detected in the deacylation products of tRNA purified from the 42S particles and from the messenger ribonucleoprotein particles (mRNPs) present in the previtellogenic oocytes of Xenopus laevis. Only a small fraction of particle tRNA carries a peptide or protein chain. The bulk of particle tRNA is simply aminoacylated. The tRNA-linked peptide chains of the particles appear to turn over more slowly in vivo than aminoacyl tRNA. These chains could arise in the particles by a peptidyl transfer reaction similar to that carried out by the ribosome.

23] Isolation of amino acid-specific tRNA by high-performance liquid chromatography

This chapter discusses the isolation of amino acid-specific transfer ribonucleic acid (tRNA) by high-performance liquid chromatography (HPCL). With new development of tRNA sequencing by the postlabeling procedure, the total nucleotide sequence of tRNA can now be determined using a very small amount of tRNA. Thus, a quick tRNA fractionation procedure is required for the purification of every small amount of tRNA. Wells and his coworkers developed reversed-phase chromatography (RPC)-5 HPLC and used it for the fractionation of DNA fragments. The chapter discusses about the commercial availability of recent HPLC columns of diethylaminoethyl (DEAE)-cellulose or RPC-5. tRNA can be fractionated much more quickly (1–2 hour) than by conventional column chromatographic procedures, and consequently a variety of pure tRNA species can be obtained within one day by use of two successive HPLCs. The chapter discusses the operation of the HPLC with several diagrams and examples.

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.

Chloroplast transfer RNAs and tRNA genes of wheat

Fractionation (by two-dimensional polyacrylamide gel electrophoresis) of total tRNA from wheat chloroplasts yields about 33 RNA spots. Of these, 30 have been identified by aminoacylation as containing tRNAs specific for 17 amino acids.Hybridization of labeled individual tRNAs to cloned chloroplast DNA fragments has revealed the location of at least nine pairs of tRNA genes in the segments of the inverted repeat, at least twelve tRNA genes in the large single copy region and one tRNA gene in the small single copy region.A comparison of this wheat chloroplast tRNA gene map to that of maize and of other higher plants suggests that gene rearrangements have occurred during evolution, even within cereal chloroplast DNA. These rearrangements have taken place within the inverted repeat, within the large single copy region and between the inverted repeat and the large single copy region.

Transfer RNA is an essential component of the ubiquitin- and ATP-dependent proteolytic system.

Protein degradation via the nonlysosomal ATP-dependent pathway in rabbit reticulocytes involves a number of components. In the initial event, ubiquitin, an abundant 76-residue polypeptide, becomes covalently linked to the protein substrate in an ATP-requiring reaction. Once marked in this way, the conjugated protein is proteolyzed in a reaction that also requires ATP. Ubiquitin-marking appears to be important to the progression of cells from one stage to another of the cell cycle; it may also be involved in gene activation. Here we show that tRNA is another essential component of the system. Ribonucleases strongly inhibit the ubiquitin- and ATP-dependent degradation of 125I-labeled bovine serum albumin in the reticulocyte system in vitro. RNAs extracted from fractions of the reticulocyte extract or from mouse cells restore proteolytic activity. When the RNA is fractionated by gel electrophoresis, only the tRNA fraction is active in restoring proteolysis. Furthermore, pure mouse tRNAHis, isolated by immunoprecipitation with patient autoimmune sera, restores the proteolytic activity. The possibility that the level of uncharged tRNA in mammalian cells regulates the ubiquitin- and ATP-dependent proteolytic system is discussed.