Rapid solid phase isolation of 20 specific tRNAs from Escherichia coli.

A solid phase procedure has been developed for the rapid isolation of all 20 species of tRNA from Escherichia coli. The overall yields for a single preparation cycle ranged from 62 to 96%, the average being 80%. The values for the amino acid acceptor activities of the tRNA species equaled those reported in the literature for highly purified tRNAs. Starting from crude tRNA, a given tRNA species can easily be isolated in less than 2 h. One milliliter of the resin, which is reusable, is sufficient for the isolation of 200 mg of a specific tRNA. The procedure requires a bifunctional reagent, one moiety of which (--SO2Cl) reacts with the amino acid on the aminoacylated tRNA, the other, with the --SH group on the resin. Thus, only the desired tRNA species is bound to the resin; any of the other tRNAs in the filtrate can be isolated in another cycle. Raising the pH results in deacylation and release from the resin of the desired tRNA species. For tRNA Cys, it is necessary to block the --SH of cysteine prior to reaction with the bifunctional reagent. Side reactions involving the bifunctional reagent. Side reactions involving the bifunctional reagent and tRNA are either easily reversible or negligible (less than 0.01%).     

Initial position of aminoacylation of individual Escherichia coli, yeast, and calf liver transfer RNAs.

Transfer RNAs from Escherichia coli, yeast (Sacharomyces cerevisiae), and calf liver were subjected to controlled hydrolysis with venom exonuclease to remove 3'-terminal nucleotides, and then reconstructed successively with cytosine triphosphate (CTP) and 2'- or 3'-deoxyadenosine 5'-triphosphate in the presence of yeast CTP(ATP):tRNA nucleotidyltransferase. The modified tRNAs were purified by chromatography on DBAE-cellulose or acetylated DBAE-cellulose and then utilized in tRNA aminoacylation experiments in the presence of the homologous aminoacyl-tRNA synthetase activities. The E. coli, yeast, and calf liver aminoacyl-tRNA synthetases specific for alanine, glycine, histidine, lysine, serine, and threonine, as well as the E. coli and yeast prolyl-tRNA synthetases and the yeast glutaminyl-tRNA synthetase utilized only those homologous modified tRNAs terminating in 2'-deoxyadenosine (i.e., having an available 3'-OH group). This is interpreted as evidence that these aminoacyl-tRNA synthetases normally aminoacylate their unmodified cognate tRNAs on the 3'-OH group. The aminoacyl-tRNA synthetases from all three sources specific argining, isoleucine, leucine, phenylalanine, and valine, as well as the E. coli and yeast enzymes specific for methionine and the E. coli glutamyl-tRNA synthetase, used as substrates exclusively those tRNAs terminating in 3'-deoxyadenosine. Certain aminoacyl-tRNA synthetases, including the E. coli, yeast, and calf liver asparagine and tyrosine activating enzymes, the E. coli and yeast cysteinyl-tRNA synthetases, and the aspartyl-tRNA synthetase from yeast, utilized both isomeric tRNAs as substrates, although generally not at the same rate. While the calf liver aspartyl- and cysteinyl-tRNA synthetases utilized only the corresponding modified tRNA species terminating in 2'-deoxyadenosine, the use of a more concentrated enzyme preparation might well result in aminoacylation of the isomeric species. The one tRNA for which positional specificity does seem to have changed during evolution is tryptophan, whose E. coli aminoacyl-tRNA synthetase utilized predominantly the cognate tRNA terminating in 3'-deoxyadenosine, while the corresponding yeast and calf liver enzymes were found to utilize predominantly the isomeric tRNAs terminating in 2'-deoxyadenosine. The data presented indicate that while there is considerable diversity in the initial position of aminoacylation of individual tRNA isoacceptors derived from a single source, positional specificity has generally been conserved during the evolution from a prokaryotic to mammalian organism.     

The effects of hyperphenylalaninaemia on the concentrations of aminoacyl-transfer ribonucleic acid in vivo. A mechanism for the inhibition of neural protein synthesis by phenylalanine.

An acute administration of phenylalanine to neonatal animals has been reported to result in large decreases in the intracellular concentrations of several essential amino acids in neural tissue, as well as an inhibition of neural protein synthesis. The present report evaluates the effects of the loss of amino acids on the concentrations of aminoacyl-tRNA in vivo, with the view that an alteration in the concentrations of specific aminoacyl-tRNA molecules could be the rate-limiting step in brain protein metabolism during hyperphenylalaninaemia. tRNA was isolated from saline- and phenylalanine-injected mice 30-45 min after injection, by using a procedure designed to maintain the concentrations of aminoacyl-tRNA present in vivo. Periodate oxidation of the non-acylated tRNA and aminoacylation with radioactively labelled amino acids was used to determine the proportion of tRNA that was present in vivo as aminoacyl-tRNA. Although decreases in the intracellular concentrations of alanine, lysine and leucine were observed after phenylalanine administration, the concentrations of alanyl-tRNA, lysyl-tRNA and leucyl-tRNA actually increased by 15%. Although tryptophan has been suggested to be rate-limiting during hyperphenylalaninaemia, the proportion of tryptophan tRNA that was acylated was maximal in both normal and hyperphenylalaninaemic animals. This unexpected increase in aminoacyl-tRNA concentration is discussed as perhaps a secondary effect resulting from the phenylalanine-induced inhibition of protein synthesis. In contrast, the proportion of methionine tRNA that was acylated in vivo after phenylalanine administration was demonstrated to be decreased by approx. 17%. When the isoaccepting species of methionine tRNA were separated by reverse-phase column chromatography, three species were separated, one of which was demonstrated to be the initiator species, tRNAfMet, by the selective aminoacylation and formylation with Escherichia coli enzymes. After the administration of phenylalanine, the acylation of each of the three methionine tRNA species was decreased, with the initiator species being lowered by 10%. This effect on aminoacylation of tRNAfMet may be the primary step by which phenylalanine affects neural protein synthesis, and this is consistent with previous reports that re-initiation may be inhibited during hyperphenylalaninaemia.     

Recognition of various arginine transfer ribonucleic acids with arginyl-tRNA synthetase purified from human placenta

Arginyl-tRNA synthetase has been purified approximately 550 fold from crude extract of human placenta by the following purification steps: Ammonium sulfate fractionation, chromatographies of DEAE-cellulose and CM-Sephadex and Sephadex G-100 gel filtration. Final preparation of this enzyme has specific activity of 123 nmole of arginyl-tRNA formed per mg of protein and was free from other aminoacyl-tRNA synthetase activities. Recognition of various arginine tRNAs with this enzyme was studied using kinetic analysis of arginylation of arginine tRNA and also arginine tRNA dependent ATP-PPi exchange reaction. Affinity of this enzyme with arginine tRNA was determine from Vmas/Km values and it was in the order of rabbit, Chum salmon, B. subtilis, E. coli and yeast in both systems.     

The nucleotide sequence of a UGA suppressor serine tRNA from Schizosaccharomyces pombe.

The UGA suppressor tRNA produced by Schizosaccharomyces pombe strain sup3-e was purified to homogeneity. It can be aminoacylated with a serine by a crude aminoacyl-tRNA synthetase preparation from S. pombe cells. By combining post-labeling fingerprinting and gel sequencing methods the nucleotide sequence of this tRNA was determined to be: pG-U-C-A-C-U-A-U-G-U-C-ac4C-G-A-G-D-G-G-D-D-A-A-G-G-A-m2G2-psi-U-A-G-A-N-U-U-C-A-i6A-A-psi-C-U-A-A-U-G-G-G-C-U-U-U-G-C-C-C-G-m5C-G-G-C-A-G-G-T-psi-C-A-m1A-A-U-C-C-U-G-C-U-G-G-U-G-A-C-G-C-C-A OH. The anticodon sequence u ca is complementary to the UGA codon.     

The identification of tRNA isoacceptors fractionated by polyacrylamide gel electrophoresis

A method is described by which specific tRNA isoacceptors may be identified in small amounts of bulk tRNA. The strategy relies on the retention of aminoacyl-tRNA by CNBr-Sepharose through covalent coupling of the alpha-NH2 group of the amino acid to the matrix. After removing unbound material by thorough washing, the bound specific isoacceptors are released by cleavage of the labile aminoacyl-tRNA ester bond through mild alkaline treatment. The product is analysed by two-dimensional gel electrophoresis and the spots obtained may be correlated with the pattern from bulk tRNA. Optimum sensitivity is achieved by combining the method with the recently introduced silver staining technique (Igloi, G.L. (1983) Anal. Biochem. 134, 184-188) for tRNA.     

Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand.

The use of tRNA affinity columns for the purification of aminoacyl-tRNA synthetases was investigated. A purification method for valyl-tRNA synthetase from Bacillus stearothermophilus is described that uses two affinity columns, one containing the pure cognate tRNA, and the other containing all tRNA species except the cognate tRNA. A method for the rapid preparation of the two columns was developed, which does not require prior isolation of cognate tRNA but makes use of the ability of the target synthetase to select its cognate tRNA. The usefulness of tRNA columns is compared with that of affinity columns derived from the aminoalkyladenylate reported in the preceding paper [Clarke & Knowles (1977) Biochem J. 167, 405-417].     

An asymmetric underlying rule in the assignment of codons: possible clue to a quick early evolution of the genetic code via successive binary choices

Aminoacyl-tRNA synthetases (aaRSs) are responsible for creating the pool of correctly charged aminoacyl-tRNAs that are necessary for the translation of genetic information (mRNA) by the ribosome. Each aaRS belongs to either one of only two classes with two different mechanisms of aminoacylation, making use of either the 2'OH (Class I) or the 3'OH (Class II) of the terminal A76 of the tRNA and approaching the tRNA either from the minor groove (2'OH) or the major groove (3'OH). Here, an asymmetric pattern typical of differentiation is uncovered in the partition of the codon repertoire, as defined by the mechanism of aminoacylation of each corresponding tRNA. This pattern can be reproduced in a unique cascade of successive binary decisions that progressively reduces codon ambiguity. The deduced order of differentiation is manifestly driven by the reduction of translation errors. A simple rule can be defined, decoding each codon sequence in its binary class, thereby providing both the code and the key to decode it. Assuming that the partition into two mechanisms of tRNA aminoacylation is a relic that dates back to the invention of the genetic code in the RNA World, a model for the assignment of amino acids in the codon table can be derived. The model implies that the stop codon was always there, as the codon whose tRNA cannot be charged with any amino acid, and makes the prediction of an ultimate differentiation step, which is found to correspond to the codon assignment of the 22nd amino acid pyrrolysine in archaebacteria.     

Evidence for the absence of the terminal adenine nucleotide at the amino acid-acceptor end of transfer ribonucleic acid in non-lactating bovine mammary gland and its inhibitory effect on the aminoacylation of rat liver transfer ribonucleic acid

1. tRNA isolated from non-lactating bovine mammary gland competitively inhibits the formation of aminoacyl-tRNA in the rat liver system. 2. Non-lactating bovine mammary gland tRNA and twice-pyrophosphorolysed rat liver tRNA are unable to accept amino acids in a reaction catalysed by aminoacyl-tRNA synthetases from either rat liver or bovine mammary gland. Deacylated rat liver tRNA can however be aminoacylated in the presence of either enzyme. 3. Bovine mammary gland tRNA lacks the terminal adenine nucleotide at the 3′-terminus amino acid acceptor end, which can be replaced by incubation in the presence of rat liver nucleotide-incorporating enzyme, ATP and CTP. 4. The enzymically modified bovine tRNA (tRNApCpCpA) can bind labelled amino acids to form aminoacyl-tRNA, which can then transfer its labelled amino acids to growing polypeptide chains on ribosomes. 5. Molecules of rat liver tRNA or bovine mammary gland tRNA that lack the terminal adenine nucleotide or the terminal cytosine and adenine nucleotides inhibit the aminoacylation of normal rat liver tRNA to varying degrees. tRNA molecules lacking the terminal −pCpCpA nucleotide sequence exhibit the major inhibitory effect. 6. The enzyme fraction from bovine mammary gland corresponding to that containing the nucleotide-incorporating enzyme in rat liver is unable to catalyse the incorporation of cytosine and adenine nucleotides in pyrophosphorolysed rat liver tRNA and deacylated bovine tRNA. This fraction also markedly inhibits the action of the rat liver nucleotide-incorporating enzyme.     

Studies on transfer RNA from mycobacteria.

Active preparations of tRNA and aminoacyl-tRNA synthetases have been isolated from exponentially growing cells of Mycobacterium smegmatis and Mycobacterium tuberculosis H37Rv. Though the aminoacyl-tRNA synthetases of older cells retain their activity, the tRNAs seem to undergo modification and show poorer activity. The mycobacterial enzyme preparations catalyse homologous and heterologous aminoacylation between tRNA from the two species (M. smegmatis and M. tuberculosis H37Rv) or from Escherichia coli, with equal efficiency; tRNA samples from eukaryotic cells (yeast and rat liver) do not serve as substrates for the mycobacterial synthetases. The analytical separation of the different amino acid specific tRNAs from M. smegmatis resembles the pattern found in other bacteria. Purification of valine- (three species) and methionine-specific tRNA (two species) to 70-80% purity has been accomplished by using column-chromatographic techniques. Of the two species of tRNAMet, one can be formylated in the presence of formyl tetrahydrofolate and the transformylase from mycobacteria.     

Knocking out a specific tRNA species within unfractionated Escherichia coli tRNA by using antisense (complementary) oligodeoxyribonucleotides

Methods for the preparation of an Escherichia coli tRNA mixture lacking one or a few specific tRNA species can be the basis for future applications of cell-free protein synthesis. We demonstrate here that virtually a single tRNA species in a crude E. coli tRNA mixture can be knocked out by an antisense (complementary) oligodeoxyribonucleotide. One out of five oligomers complementary to tRNA^Asp blocked the aspartylation almost completely, while minimally affecting the aminoacylation with other 13 amino acids tested. This `knockout' tRNA behaved similarly to the untreated tRNA in a cell-free translation of an mRNA lacking Asp codons.     

Rates of aminoacyl-transfer-ribonucleic acid synthesis in vivo and in vitro by bean leaves

1. A procedure for measuring rates of aminoacyl-tRNA synthesis in vitro and in intact leaves is presented. 2. Leaf discs showed rates close to those of intact leaves. 3. Cell-free preparations showed similar rates when assayed by pyrophosphate exchange, but actual aminoacyl-tRNA formation rates appeared to be much lower. Evidence is presented that dilution of supplied labelled amino acids was a major factor causing the low apparent rates. 4. Attempts to strip endogenous amino acids from plant tRNA resulted in low acceptor capability of the tRNA.     

Study of the photoaffinity modification of Escherichia coli ribosomes near the donor tRNA-binding center

Affinity labelling of E. coli ribosomes near the donor tRNA-binding (P) site was studied with the use of photoreactive derivatives of tRNAPhe bearing arylazidogroups on N7 atoms of guanine residues (azido-tRNA). UV-irradiation of complexes 70S ribosome.poly(U).azido- tRNA(P-site) and 70S ribosome.poly(U).azido-tRNA(P-site).Phe- tRNAPhe(A-site) resulted in covalent attachment of azido-tRNA to ribosomes, both subunits being labelled. In both cases modification extent of 30S subunit was two-fold than that of the 50S one. It was shown that when the A-site was free the azido-tRNA located in P-site labelled proteins S9, S11, S12, S13, S21 and L14, L27, L31. Azido-tRNA located in P-site when the A-site was occupied with Phe-tRNAPhe labelled proteins S11, S12, S13, S14, S19, L32/L33 and possibly L23, L25. From the comparison of the sets of proteins labelled when A-site was free or occupied a conclusion was drawn that aminoacyl-tRNA located in ribosomal A-site affects the arrangement of deacylated tRNA in P-site. Data obtained allow to propose that proteins S5, S19, S20 and L24, L33 interact with guanine residues important for the tRNA tertiary structure formation.     

Mitochondrially-imported cytoplasmic tRNA(Lys)(CUU) of Saccharomyces cerevisiae: in vivo and in vitro targetting systems.

The cytoplasmic tRNA(Lys)(CUU) (tRNA(1Lys)) is the single yeast tRNA species to be traffiked from the cytoplasm into the mitochondrial compartment of the cell. To study mechanisms of this targetting we worked out two test systems. The in vivo system based on the electroporation of intact yeast cells was used to introduce labelled tRNAs into the cytoplasm. All tRNA species tested were effectively introduced into the cytoplasm, but only the cytoplasmic tRNA(1Lys) was found in the mitochondrial compartment within 1-2 hours after the electroporation procedure. The in vitro system permits specific transfer of the tRNA(1Lys) into isolated mitochondria. Contrary to the known systems for protein transport into isolated mitochondria, mitochondrial import of tRNA(1Lys) in vitro requires the presence of soluble cellular proteins in the reaction mixture. The translocation proved to be ATP-dependent and to require the presence of an ATP-generation system in the reaction. Preincubation of the tRNA with the total cellular extract of the cell markedly increases the rate of the translocation. Two protein fractions are necessary to direct the import in vitro. The first one has high heparin-binding affinity, while the other protein fraction is not retained by heparin-Sepharose.     

Transfer RNA aminoacylation: identification of a critical ribose 2'-hydroxyl-base interaction.

To understand the relationship between tRNA architecture and specific aminoacylation by aminoacyl-tRNA synthetases, we performed kinetic assays of Escherichia coli tRNA(Pro) molecules containing single deoxynucleotide substitutions. We identified an important 2'-hydroxyl group at position U8 (of 22 positions probed). Chemical modification studies showed that this 2'-hydroxyl interacts with either the N1 or the exocyclic amine of G46 in a hydrogen bonding interaction that contributes 1.8 kcal/mol to the free energy of activation for aminoacylation. Molecular modeling of tRNA(Pro) supports the existence of this interaction. This is the first study to identify a specific ribose 2'-hydroxyl-base interaction in the core region of a tRNA molecule that makes a thermodynamically significant contribution to aminoacylation.     

Fractionation of specific mitochondrial and cytoplasmic tRNAs obtained from calf brain.

—Total tRNA fractions were isolated from pure mitochondrial and cytoplasmic calf brain preparations. After incubation with homologous crude preparations of aminoacyl-tRNA ligases in the presence of [¹⁴C]-glutamic acid, tRNAs were separated chromatographically on BD-cellulose columns and in reversed phase chromatography systems. In both of the methods used, cytoplasmic tRNA preparations revealed a larger number of radioactivity peaks. In experiments with double labelling, five radioactivity peaks for cytoplasmic glutamyl-tRNAs corresponded to only three mitochondrial glutamyl-tRNA fractions. The results imply the presence of isoaccepting species of tRNA in brain.     

Identification of an apparent aminoacyl-tRNA synthetase activator factor as tRNA nucleotidyltransferase

Summary The ability of yeast extracts to aminoacylate crude yeast tRNA with leucine and other amino acids is largely lost after chromatography of the extracts in DEAE-Sephadex. The original aminoacylating ability is restored by combining protein fractions from the DEAE-chromatogram. The characteristics of this reactivation are very similar to the activation, by protein factors, of certain aminoacyl-tRNA synthetases reported by others. The results in this work indicate that the apparent aminoacyl-tRNA synthetase activator factor is the tRNA nucleotidyltransferase and that the restoration of the original tRNA aminoacylating ability is a consequence of the repairing of the 3' end of incomplete tRNA chains.