Calf Liver tRNA Research Articles

Interaction of Immobilized Human Exportin-t with Calf Liver tRNA

The binding of tRNA species from calf liver to immobilized exportin-t in the presence of ran•GppNHp was examined by affinity chromatography. We observed different eluting behaviors of individual tRNAs. After separating tRNAs on a two-dimensional polyacrylamide gel, the positions of 7 selected tRNAs were identified by Northern hybridization and their relative affinities to immobilized exportin-t•ran•GppNHp complex were estimated in the order of tRNALeuCAG > tRNASerGCU, tRNALeuCAA, tRNASerUGA > tRNASerAGA, tRNALeuAAG > tRNAArgACG. We propose that exportin-t preferentially binds and exports those tRNAs that are rapidly consumed by the protein synthesis machinery.

The C-terminal Appended Domain of Human Cytosolic Leucyl-tRNA Synthetase Is Indispensable in Its Interaction with Arginyl-tRNA Synthetase in the Multi-tRNA Synthetase Complex

Human cytosolic leucyl-tRNA synthetase is one component of a macromolecular aminoacyl-tRNA synthetase complex. This is unlike prokaryotic and lower eukaryotic LeuRSs that exist as free soluble enzymes. There is little known about it, since the purified enzyme has been unavailable. Herein, human cytosolic leucyl-tRNA synthetase was heterologously expressed in a baculovirus system and purified to homogeneity. The molecular mass (135 kDa) of the enzyme is close to the theoretical value derived from its cDNA. The kinetic constants of the enzyme for ATP, leucine, and tRNA(Leu) in the ATP-PP(i) exchange and tRNA leucylation reactions were determined, and the results showed that it is quite active as a free enzyme. Human cytosolic leucyl-tRNA synthetase expressed in human 293 T cells localizes predominantly to the cytosol. Additionally, it is found to have a long C-terminal extension that is absent from bacterial and yeast LeuRSs. A C-terminal 89-amino acid truncated human cytosolic leucyl-tRNA synthetase was constructed and purified, and the catalytic activities, thermal stability, and subcellular location were found to be almost identical to native enzyme. In vivo and in vitro experiments, however, show that the C-terminal extension of human cytosolic leucyl-tRNA synthetase is indispensable for its interaction with the N-terminal of human cytosolic arginyl-tRNA synthetase in the macromolecular complex. Our results also indicate that the two molecules interact with each other only through their appended domains.

In Vitro Evidence That the Untranslated Leader of the HIV-1 Genome Is an RNA Checkpoint That Regulates Multiple Functions through Conformational Changes

The HIV-1 RNA genome forms dimers through base pairing of a palindromic 6-mer sequence that is exposed in the loop of the dimer initiation signal (DIS) hairpin structure (loop-loop kissing). The HIV-1 leader RNA can adopt a secondary structure conformation that is not able to dimerize because the DIS hairpin is not folded. Instead, this DIS motif is base-paired in a long distance interaction (LDI) that extends the stem of the primer-binding site domain. In this study, we show that targeting of the LDI by either antisense oligonucleotides or specific mutations can induce the conformational switch to a branched multiple hairpin (BMH) structure, and this LDI-to-BMH switch coincides with increased RNA dimerization. Another interesting finding is that the extended LDI stem can resist a certain level of destabilization, indicating that a buffer is created to prevent a premature conformational switch and early dimerization. Because the tRNA(Lys3) primer for reverse transcription anneals to multiple sequence elements of the HIV-1 leader RNA, including sequences in the LDI stem, we tested whether tRNA-annealing can destabilize the LDI stem such that RNA dimerization is triggered. Using a combination of stem-destabilizing approaches, we indeed measured a small but significant effect of tRNA-annealing on the ability of the RNA template to form dimers. This observation suggests that HIV-1 RNA can act as a checkpoint to control and coordinate different leader functions through conformational switches. This in vitro result should be verified in subsequent in vivo studies with HIV-infected cells.

In Vitro Studies on tRNA Annealing and Reverse Transcription with Mutant HIV-1 RNA Templates

The human immunodeficiency virus type 1 (HIV-1) RNA genome encodes a semistable stem-loop structure, the U5-PBS hairpin, which occludes part of the tRNA primer binding site (PBS). In previous studies, we demonstrated that mutations that alter the stability of the U5-PBS hairpin inhibit virus replication. A reverse transcription defect was measured in assays with the virion-extracted RNA-tRNA complexes. We now extend these studies with in vitro synthesized wild-type and mutant RNA templates that were tested in primer annealing and reverse transcription assays. The effect of annealing temperature and the presence of the viral nucleocapsid protein on reverse transcription was analyzed for the templates with a stabilized or destabilized U5-PBS hairpin, and in reactions initiated by tRNA or DNA primers. The results of this in vitro assay are consistent with the in vivo findings, in that both tRNA annealing and initiation of reverse transcription are sensitive to stable template RNA structure. Reverse transcription initiated by a DNA primer is less hindered by secondary structure in the RNA template than tRNA primed reactions. The inhibitory effect of template structure on tRNA-primed reverse transcription is more pronounced in this in vitro assay compared with the in vivo material, indicating that the heat-annealed RNA-tRNA complex differs from the virion-extracted viral RNA-tRNA complex.

Yeast Asparagine (Asn) tRNA without Q Base Promotes Eukaryotic Frameshifting More Efficiently than Mammalian Asn tRNAs with or without Q Base

In this study, we compare the efficiency of Asn tRNA from mammalian sources with and without the highly modified queuosine (Q) base in the wobble position of its anticodon and Asn tRNA from yeast, which naturally lacks Q base, to promote frameshifting. Interestingly, no differences in the ability of the two mammalian Asn tRNAs to promote frameshifting were observed, while yeast tRNAAsn −Q promoted frameshifting more efficiently than its mammalian counterparts in both rabbit reticulocyte lysates and wheat germ extracts. The shiftability of yeast Asn tRNA is therefore not due, or at least not completely, to the lack of Q base and most likely the shiftiness resides in structural differences elsewhere in the molecule. However, we cannot absolutely rule out a role of Q base in frameshifting as wheat germ extracts and a lysate depleted of most of its tRNA and supplemented with calf liver tRNA contain both Asn tRNA with or without Q base.

Genomic organization, cDNA sequence, bacterial expression, and purification of human seryl-tRNA synthase.

In this paper, we report the cDNA sequence and deduced primary sequence for human cytosolic seryl-tRNA synthetase, and its expression in Escherichia coli. Two human brain cDNA clones of different origin, containing overlapping fragments coding for human seryl-tRNA synthetase were sequenced: HFBDN14 (fetal brain clone); and IB48 (infant brain clone). For both clones the 5' region of the cDNA was missing. This 5' region was obtained via PCR methods using a human brain 5' RACE-Ready cDNA library. The complete cDNA sequence allowed us to define primers to isolate and characterize the intron/exon structure of the serS gene, consisting of 10 introns and 11 exons. The introns' sizes range from 283 bp to more than 3000 bp and the size of the exons from 71 bp to 222 bp. The availability of the gene structure of the human enzyme could help to clarify some aspects of the molecular evolution of class-II aminoacyl-tRNA synthetases. The human seryl-tRNA synthetase has been expressed in E. coli, purified (95% pure as determined by SDS/PAGE) and kinetic parameters have been measured for its substrate tRNA. The human seryl-tRNA synthetase sequence (514 amino acid residues) shows significant sequence identity with seryl-tRNA synthetases from E. coli (25%), Saccharomyces cerevisiae (40%), Arabidopsis thaliana (41%) and Caenorhabditis elegans (60%). The partial sequences from published mammalian seryl-tRNA synthetases are very similar to the human enzyme (94% and 92% identity for mouse and Chinese hamster seryl-tRNA synthetase, respectively). Human seryl-tRNA synthetase, similar to several other class-I and class-II human aminoacyl-tRNA synthetases, is clearly related to its bacterial counterparts, independent of an additional C-terminal domain and a N-terminal insertion identified in the human enzyme. In functional studies, the enzyme aminoacylates calf liver tRNA and prokaryotic E. coli tRNA.

Gene Mapping and Expression of Tomato Bushy Stunt Virus

Summary Cloned DNA species complementary to the RNA of tomato bushy stunt virus (TBSV) were produced and the location of each on the virus genome was determined. Polysomes were prepared from TBSV-infected plants and RNA, released from the polysomes by treatment with puromycin, was shown by Northern hybridization to contain TBSV genomic RNA (4.8 kb) and two subgenomic ssRNA species of 2.0 and 0.9 kb. Hybrid selection using clones corresponding to different parts of the virus genome showed that the two subgenomic RNA species are probably 3′ coterminal with the genomic RNA. In vitro translation of hybrid-selected RNA showed that the genomic (4.8 kb), 2.0 and 0.9 kb RNA species encode proteins of M r 37000 (37K), 40K (the virus coat protein) and 22K respectively. Translation in the presence of calf liver tRNA produced, in addition to the other products, a protein of M r 90K which may be a readthrough product of the 37K protein. The results have enabled a model for the genome organization and expression of TBSV to be formulated.

Ribosomes are stalled during in vitro translation of alfalfa mosaic virus RNA 1

In the presence of plant tRNAs the full-length translation product of alfalfa mosaic virus RNA 1 is produced in rabbit reticulocytes only at low mRNA concentration. At higher mRNA concentration translation is restricted to the 5' half of RNA 1. At high mRNA concentration the full-length product can be formed when additional plant tRNA and glutamine are supplied to the translation mixture. In contrast, in the presence of yeast or calf liver tRNA the translation pattern of alfalfa mosaic virus RNA 1 always results in the synthesis of the full-length product. Pulse-chase experiments in the presence of plant tRNAs show that the ribosomes pause at several positions in the 5' half of RNA 1. The pausing time is different at the different 'halting places'. Protein synthesis is resumed upon addition of glutamine, even when the addition is delayed for more than 3 h after the start of protein synthesis. Only one tRNA species, purified from wheat germ or tobacco, could promote full-length translation of RNA 1. This tRNA can be charged with glutamine. Analysis of the position of glutamine codons on RNA 1 shows a correlation between the positions of the CAA codons and the halting places of the ribosomes. The CAA codon (for any other codon) on its own cannot be responsible for the pausing of the ribosomes, since a variety of RNAs, known to contain all sense codons, are translated efficiently in rabbit reticulocyte lysates in the presence of plant tRNAs. Apparently other elements can restrict decoding of normal codons during protein chain elongation.

The effects of deacylated transfer ribonucleic acid on polypeptide-chain initiation in Ehrlich-ascites-tumour-cell extracts [proceedings

In intact Ehrlich ascites-tumour cells the rate of protein synthesis is decreased by over 6096 in the absence of a single essential amino acid (van Venrooij et al., 1972). This effect is due largely to an inhibition of polypeptide-chain initiation, and is associated with polyribosome disaggregation and a decrease in the number of Met-tRNAme-40 S subunit initiation complexes (Pain & Henshaw, 1975). Cell-free extracts prepared from control and lysinedeprived cells show some of the characteristics of the cells from which they were derived. Protein synthesis in starved-cell extracts is decreased by up to 75% relative to that in fed-cell extracts. Starved-cell extracts also have a diminished ability to form initiation complexes between 40s subunits and Met-tRNAme. This defect is abolished by addition of highly purified initiation factor eIF-2 (Pain et al., 1980; Clemens et al., 1980). It has been suggested (Vaughan & Hansen, 1973; Kyner el al., 1973) that the inhibition of polypeptide-chain initiation seen in cells deprived of amino acids may be due to increases in the concentrations of deacylated tRNA species. To test whether. increased concentrations of deacylated tRNA could directly inhibit polypeptide-chain initiation, the effect of adding periodate-oxidized tRNA (which cannot be charged with amino acids) to cell-free extracts from Ehrlich ascites-tumour cells was examined. Cell-free extracts from Ehrlich ascites-tumour cells were prepared, and analysis of labelled initiation complexes by sucrose-gradient centrifugation was performed as described by Pain et al. (1980). Deacylated calf liver tRNA was subjected to periodate oxidation as described by Vaughan & Hansen (1973). Addition of deacylated oxidized tRNA to cell-free extracts From Ehrlich ascites-tumour cells in the presence of 1 mMemetine to prevent polypeptide-chain elongation causes only a 10-2096 decrease in the concentration of Met-tRNAmet-40 S subunit initiabon complexes, at concentrations between 20 and 1OOpg of added tRNA/ml (Table 1). This small inhibition is not concentration-dependent. A much greater effect is seen on Met-tRNAme-80S ribosome initiation-complex formation. Addition of 20pg of deacylated tRNA/ml causes an 85% decrease in the concentration of Met-tRNAMet-80 S ribosome complexes. At higher concentrations of added tKNA Met-tRNAmet-80S ribosome complexes are completely abolished (Table 1). Addition of deacylated oxidized tRNA to cell-free extracts in the absence of emetine, where protein synthesis is occurring, causes an accumulation of Met-tRNAme-40S subunit initiation complexes. This implies impairment of a stage after MettRNAWCt-40S subunit initiation-complex formation, possibly 40s subunit-60s subunit joining. However, h this situation the concentrations of complexes seen just reflect the balance between their rate of formation and their rate of utilization, and the results are more difficult to interpret than those of experiments performed in the presence of an inhibitor of elongation. We have shown that concentrations of deacylated oxidized tRNA in the range 20-100pg/ml cause only a small decrease in the Concentration of Met-tRNAwd-40S subunit initiationcomplex formation. Since the concentration of Met-tRNAme40s subunit initiation complexes in extracts from lysinedeprived cells is decreased by up to 77% compared with that in extracts from control cells (Pain et al., 1980; Clemens et al., 1980), the small decline in initiation complexes seen with deacylated tRNA is not enough to account for the differences between fed-cell and starved-cell systems. It would seem therefore that an increase in the concentration of deacylated tRNA after amino acid deprivation is not the direct cause of the inhibition of this step of polypeptide-chain initiation in Ehrlich ascitestumour cells under these circumstances. Deacylated tRNA is strongly inhibitory to Met-tRNAmet-80s ribosome initiation-complex formation. This effect may be due to impairment of the conversion of Met-tRNAm-40S subunit initiation complexes into Met-tRNAmet-80S ribosome initiation complexes or to a destabilization of the latter complexes. The accumulation of Met-tRNAmet40S subunit initiation complexes seen in the presence of deacylated tRNA under protein-synthesis conditions is consistent with the former model. These results are also in agreement with the findings obtained by Kyner et al. (1973), who reported an inhibition of Met-tRNAmd-80S ribosome initiation-complex formation by uncharged tRNA in a fractionated cell-free system. As deacylated tRNA is unlikely to be directly responsible for the inhibition of polypeptide-chain initiation at the stage of Met-tRNAme40s subunit initiation-complex formation, the mechanism by which this part of the protein-synthesizing machinery of Ehrlich ascites-tumour cells recognizes the lack of one amino acid remains to be elucidated. This work is supported by grants from the Medical Research Council and the Cancer Research campaign. Clemens, M. J., Henshaw, E. C. & Pain, V. M. (1980) Blochem. Soc. Trans. 8,350-351

In Vitro translation of immediate early, early, and late classes of RNA from vaccinia virus-infected cells

Cytoplasmic RNA, isolated at various times after vaccinia virus infection, was translated in a message-dependent cell-free system prepared from rabbit reticulocytes. Supplementation of the system with calf liver tRNA specifically increased translation of viral RNA. Virtually all of the [ 35S]methionine-labeled viral proteins from infected cells that were detected by sodium dodecyl sulfate -polyacrylamide gel electrophoresis appeared to be synthesized in the cell-free system. When programmed with RNA extracted at 2 hr after infection, early viral proteins were made and formation of cellular proteins was diminished. Primarily late proteins were synthesized using RNA extracted at 4 or more hr after infection, suggesting that the switch in protein synthesis is regulated principally by changes in RNA concentration rather than by modification of the translation apparatus of the cell. However, the vaccinia virus-mediated inhibition of host protein synthesis that occurred in the presence of actinomycin D was not associated with a decrease in translatable cellular mRNA. Immediate early RNA and early RNA were obtained by infecting cells in the presence of inhibitors of protein and of DNA synthesis, respectively. Analysis of the in vitro translation products did not reveal a class of early genes that require protein synthesis for expression. On the contrary, seven polypeptides, of which a 28,000-dalton species was most prominent, were synthesized in relatively greater amounts with immediate early RNA than with early RNA. All early and late mRNA species appear to be polyadenylylated, and a correlation between RNA sedimentation and molecular weight of translation product was obtained.

Incorporation of lysine into Y base of phenylalanine tRNA in Vero cells

Vero cells, a line derived from African green monkey kidney, contains a hypermodified base, called Y, adjacent to the 3' end of the anticodon of tRNAPhe. Two types of evidence are presented suggesting that lysine is involved in biosynthesis of Y base in these cells. First, when Vero cells are starved for lysine, a new, early-eluting species of tRNAPhe which lacks the fully modified Y base can be detected by reversed phase chromatography (RPC-5). After addition of lysine to the medium, this new species disappears. Second, when these cells are grown in low-lysine medium and then exposed to [3H]lysine, radioactivity from the lysine comigrates with tRNAPhe. The Y base can be selectively excised from tRNAPhe by incubation at pH 2.9, and extracted into ethyl acetate. Thin-layer chromatography of acid-excised material from these cells reveals that lysine-derived radioactivity comigrates with genuine Y base from calf liver tRNAPhe and the acid-excised tRNA no longer contains radioactivity. These results are consistent with the model that lysine is a structural precursor of Y base in tRNAPhe of Vero cells.

Sequence studies on tRNA Phe from human placenta: Comparison with known sequences of tRNA Phe from other normal mammalian tissues

Phenylalanine-specific tRNA was isolated from human placenta and degraded to mixtures of oligonucleotides. Tritium sequence analysis of the digestion products indicates that the sequence of human placenta tRNA Phe is identical to that of calf liver tRNA Phe and differs only slightly from that of rabbit liver tRNA Phe.

Preparation of crude transfer RNA and chromatographic purification of five transfer RNAs from calf liver

Five tRNAs (aspartic acid-2, leucine-1, lysine-2, phenylalanine, and serine-3) were isolated from crude calf liver tRNA by chromatographic procedures involving benzoylated DEAE-cellulose, reversed-phase chromatography, and DEAE-Sephadex A-50. Calculated activities (amino acid acceptance per 3′-terminal adenosine) were from 87 to 102 %. Crude calf liver tRNA contains much smaller quantities of RNA contaminants than crude tRNA from the mature beeves. The procedures for the preparation of the crude tRNA and chromatographic steps for isolation of these five tRNAs are amenable to scale-up for the preparation of larger samples.

Columns for rapid chromatographic separation of small amounts of tracer-labeled transfer ribonucleic acids

A small reversed-phase chromatographic system for the separation of [ 3H] or [ 14C] aminoacyl-tRNA's is described. This system has the advantages of speed (chromatographic runs can be completed in 30 to 60 min), sensitivity (samples containing as little as 3.5 × 10 −9 gm or 60 × 16 −6 A 260 unit of phenylalanyl-tRNA have been resolved as a chromatographic peak), and excellent resolution of isoaccepting tRNA's. The system has been applied to samples of E. coli tRNA's and calf liver tRNA's.

Formula omitted] studies of protein synthesis in Drosophila

Comparative in, vitro studies of protein synthesis in Oregon-R wild-type and A 53g mutant Drosophila, melanogaster indicate that the A 53g genotype has an effect at the translation level. The A 53g post-microsomal supernatant fluid stimulates the incorporation of C 14 amino acids by cell-free systems using ribosomes prepared from either A 53g or Oregon-R wild-type flies. The level of in, vitro charging of Drosophila and calf liver tRNA with C 14 amino acids by the A 53g post-microsomal supernatant fraction is also greater than the level of charging by the Oregon-R supernatant fluid.