Position Of tRNA Research Articles

Coding properties of 5-methioxyuridine in the wobble position of threonine and valine tRNAs from Bacillus subtilis.

The coding properties of 5-methoxyuridine in the wobble position of threonine and valine tRNAs from Bacillus subtilis were investigated by the technique of aminoacyl-tRNA binding to ribosomes. 5-Methoxyuridine is recognized by A, G, and fairly well by U of the third letter of colons like uridine-5-oxyacetic acid in some tRNAs from Escherichia coli.

Absence of tRNA-guanine transglycosylase in a human colon adenocarcinoma cell line

Queuosine (Q), found exclusively in th first position of the anticodons of tRNA Asp, tRNA Asn, tRNA His and tRNA Tyr, is synthesized in eucaryotes by a base-for-base exchange of queuine, the base of Q, for guanine at tRNA position 34. This reaction is catalyzed by the enzyme tRNA-guaning transglucosylate (EC 2.4.2.29). We measured the specific release of queuine from Q-5′-phsphate (queuine salvage) and the extent of tRNA Q modification in 6 human tumors carried as xenografts in immune-deprived mice. Q-deficient tRNA was found in 3 of the tumors but it did not correlate with diminished queuine salvage. The low tRNA Q content of one tumor, the HxGC 3 colo adenocarcinoma, prompted us to examine a HxGC 3-derived cell line, GC 3/M. GC 3/M completely lacks Q in its tRNA and measurable tRNA-guanine transglycosylase activity; the first example of a higher eucaryotic cell which lacks this enzyme. Exposure of GC 3/M cells to 5-azacytidine induces the transient appearance of Q-positive tRNA. This result suggests that at least one allele of the transglycosylase gene in GC 3/M cells may have been inactivated by DNA methylation. In clinical samples, we found Q-deficient tRNA in 10 of 46 solid tumors, including 2 of 13 colonic carcinomas.

In vitrostudy ofE.colitRNAArgand tRNALysidentity elements

Various tRNA transcripts were constructed to study the identity elements of E.coli tRNA(Arg) and tRNA(Lys). Exchange of the anticodon of the major tRNA(Arg) from ACG to either CCG or CCU did not result in a significant loss of arginine acceptor activity, whereas not only that to UUU but also that to ACA or ACC decreased the activity. Base substitutions and deletion at A20 also impaired the arginine charging activity by over 50-fold. Arginine charging activity was introduced by either substitution of the anticodon from UAC to ACG in tRNA(Val) or from UUU to UCU in tRNA(Lys). Only a single base substitution at the third position of tRNA(Trp) anticodon (CCA) from A to G also gave rise to arginine charging activity, which was elevated to a comparable level to that of the tRNA(Arg) transcript by an additional A20 insertion. Base substitutions of the major tRNA(Arg) at the discriminator position into pyrimidines led to a decrease by factors of three to four. These data show that the third letter of the anticodon G36 or U36 besides the second letter C35 and the A20 in the variable pocket is responsible for the arginine acceptor identity, to which the discriminator base A73 or G73 contributes in an auxiliary fashion. In contrast to the arginine system, the transcript with the wild-type tRNA(Lys) sequence showed only 140-fold lower lysine charging activity than the native tRNA(Lys), suggesting the involvement of base modifications in recognition. Replacement of the anticodon UUU with not only UCU and UAC but also UUA and UUC seriously affected the lysine acceptor activity, and those with GUU and UUG also decreased by factors of 17 and 5, respectively. Introduction of UUU into the anticodons conferred lysine charging activity upon both tRNA(Val) and tRNA(Arg). Substitution of the discriminator base A73 by any of the other bases decreased the lysine acceptor activity by a factor of ten. These results indicate the involvements of all the three bases of the anticodon and A at the discriminator position in lysine specific aminoacylation.

Competition of aminoacyl-tRNA synthetases for tRNA ensures the accuracy of aminoacylation.

The accuracy of protein biosynthesis rests on the high fidelity with which aminoacyl-tRNA synthetases discriminate between tRNAs. Correct aminoacylation depends not only on identity elements (nucleotides in certain positions) in tRNA (1), but also on competition between different synthetases for a given tRNA (2). Here we describe in vivo and in vitro experiments which demonstrate how variations in the levels of synthetases and tRNA affect the accuracy of aminoacylation. We show in vivo that concurrent overexpression of Escherichia coli tyrosyl-tRNA synthetase abolishes misacylation of supF tRNA(Tyr) with glutamine in vivo by overproduced glutaminyl-tRNA synthetase. In an in vitro competition assay, we have confirmed that the overproduction mischarging phenomenon observed in vivo is due to competition between the synthetases at the level of aminoacylation. Likewise, we have been able to examine the role competition plays in the identity of a non-suppressor tRNA of ambiguous identity, tRNA(Glu). Finally, with this assay, we show that the identity of a tRNA and the accuracy with which it is recognized depend on the relative affinities of the synthetases for the tRNA. The in vitro competition assay represents a general method of obtaining qualitative information on tRNA identity in a competitive environment (usually only found in vivo) during a defined step in protein biosynthesis, aminoacylation. In addition, we show that the discriminator base (position 73) and the first base of the anticodon are important for recognition by E. coli tyrosyl-tRNA synthetase.

Competition of aminoacyl-tRNA synthetases for tRNA ensures the accuracy of aminoacylation

The accuracy of protein biosynthesis rests on the high fidelity with which aminoacyl-tRNA synthetases discriminate between tRNAs. Correct aminoacylation depends not only on identity elements (nucleotides in certain positions) in tRNA (1), but also on competition between different synthetases for a given tRNA (2). Here we describe in vivo and in vitro experiments which demonstrate how variations in the levels of synthetases and tRNA affect the accuracy of aminoacylation. We show in vivo that concurrent overexpression of Escherichia coli tyrosyl-tRNA synthetase abolishes misacylation of supF tRNA(Tyr) with glutamine in vivo by overproduced glutaminyl-tRNA synthetase. In an in vitro competition assay, we have confirmed that the overproduction mischarging phenomenon observed in vivo is due to competition between the synthetases at the level of aminoacylation. Likewise, we have been able to examine the role competition plays in the identity of a non-suppressor tRNA of ambiguous identity, tRNA(Glu). Finally, with this assay, we show that the identity of a tRNA and the accuracy with which it is recognized depend on the relative affinities of the synthetases for the tRNA. The in vitro competition assay represents a general method of obtaining qualitative information on tRNA identity in a competitive environment (usually only found in vivo) during a defined step in protein biosynthesis, aminoacylation. In addition, we show that the discriminator base (position 73) and the first base of the anticodon are important for recognition by E. coli tyrosyl-tRNA synthetase.

5-[[(Carboxymethyl)amino]methyl]uridine is found in the anticodon of yeast mitochondrial tRNAs recognizing two-codon families ending in a purine

The modified nucleoside (U*) present in the wobble position of Saccharomyces cerevisiae mitochondrial tRNA(Leu) and tRNA(Trp) was isolated by thin-layer chromatography and HPLC. Its chromatographic, UV spectral, and mass spectrometric properties were shown to be identical with those of 5- [[(carboxymethyl)amino]methyl]uridine (cmnm5U). This nucleoside found in yeast mitochondrial tRNAs reading two-codon families ending in a purine permits the selective recognition of A and G in the third codon position.

Chorismic acid, a key metabolite in modification of tRNA

Chorismic acid is the common precursor for the biosynthesis of the three aromatic amino acids as well as for four vitamins. Mutants of Escherichia coli defective in any of the genes involved in the synthesis of chorismic acid are also unable to synthesize uridine 5-oxyacetic acid (cmo5U) and its methyl ester (mcmo5U). Both modified nucleosides are normally present in the wobble position of some tRNA species. Mutants defective in any of the specific pathways leading to phenylalanine, tyrosine, tryptophan, folate, enterochelin, ubiquinone, and menaquinone have normal levels of cmo5U and mcmo5U in their tRNA. The presence of shikimic acid in the growth medium restores the ability of an aroD mutant to synthesize cmo5U, while O-succinylbenzoate, which is an early intermediate in the synthesis of menaquinone, does not. Thus, chorismic acid is a key metabolite in the synthesis of these two modified nucleosides in tRNA. The absence of chorismic acid blocks the formation of cmo5U and mcmo5U at the first step, which might be the formation of 5-hydroxyuridine. This results in an unmodified U in the wobble position of tRNA(1Val) and in most of the tRNAs normally containing cmo5U and mcmo5U. Since cmo5U and mcmo5U are synthesized under anaerobic conditions, the formation of these nucleosides does not require molecular oxygen. One of the carbon atoms of the side chain, --O--CH2--COOH, originates from the methyl group of methionine. The other carbon atom does not originate directly from the C-1 pool, from the carboxyl group methionine, or from bicarbonate. This metabolic link between intermediary metabolism and translation also exists for another member of the family Enterobacteriaceae, Salmonella typhimurium, as well as for the distantly related gram-positive organism Bacillus subtilis.

Codon discrimination and anticodon structural context.

Site-directed mutagenesis has been used to change the nucleotide C in the wobble position of tRNA(1Gly) (CCC) to U. The mutated tRNA was tested for its ability to read glycine codons in an in vitro protein-synthesizing system programmed with the phage message MS2-RNA that had been modified by site-directed mutagenesis so as to make it possible to monitor conveniently the reading of all four glycine codons. The results showed that while the efficiency of tRNA(1Gly) (UCC) was comparable to that of mycoplasma tRNA(Gly) (UCC) in the reading of the codon GGA, the mycoplasma tRNA(Gly) was far more efficient than the tRNA(1Gly) (UCC) in the reading of the codons GGU and GGC. Thus, the anticodon UCC, when present in the structural context of the tRNA(1Gly) molecule, behaved as predicted by the wobble rules while in the structural context of the mycoplasma tRNA(Gly) it read without discrimination between the nucleotides in the third codon position, in violation of the wobble restrictions. The result with the codon GGG showed that the anticodon UCC, when present in tRNA(1Gly), was considerably less efficient in reading this codon than it was in the structural context of the mycoplasma tRNA(Gly). It would therefore seem that the anticodon UCC, when present in a certain tRNA, can be an efficient wobbler, while in the molecular environment of another tRNA it is markedly restricted in its ability to wobble.

An efficiently transcribed human tRNA(Val) gene variant produces a stable pre-tRNA: repair of the processing defect by in vitro mutations.

A tRNA(CACVal) gene variant, pHtV4, was cloned from human placenta genomic DNA. This gene differs from a closely related, functional tRNA(CACVal) gene by four base exchanges: T residues in place of C25, C62, and C66 create G:U pairs, and an A instead of G65 creates an A:C mismatch in the corresponding RNA transcript. The tRNA(Val) gene variant in pHtV4 is efficiently transcribed in HeLa cell nuclear extracts; however, the resulting pre-tRNA is processing-deficient, i.e., neither its 5'- nor its 3'-flanking sequences are removed to generate mature tRNA. Reversion of all four point mutations in pHtV4 by oligonucleotide-directed mutagenesis yielded a functional tRNA(CACVal) gene within the flanks of pHtV4, the pre-tRNA of which was processed to mature tRNA. Construction of a chimeric tRNA(Val) gene and site-directed mutagenesis of the tRNA(Val) gene in pHtV4, respectively, followed by transcription and processing studies showed that each of the four mutations contributes to the processing defect of the pHtV4-derived pre-tRNA. Moreover, this revealed that G:U pairs, which are common in all tRNAs, can impair pre-tRNA processing and therefore do not occur in certain positions in eukaryotic tRNAs.

25] Affinity labeling of tRNA-binding sites on ribosomes

During the past several years a variety of chemically reactive derivatives of tRNA have been synthesized for use as affinity labels for amino acid-tRNA ligases, ribosomes, and factors involved in protein biosynthesis. Particularly in the study of Escherichia coli ribosomes, progress has been rather rapid. Besides affinity-label derivatives of tRNA, reactive derivatives of oligo- and polynucleotides have been constructed to label the ribosomal site for mRNA binding. Furthermore, a number of small-molecular-weight substances, such as antibiotics and GTP, have been chemically modified to obtain specific reagents for their respective ribosomal binding sites. The field of affinity labeling of ribosomes has been reviewed before with emphasis on various aspects of this problem (Cantor et al. 1974; Pellegrini and Cantor 1977; Zamir 1977; Cooperman 1978; Kuechler 1978). Here we want to concentrate primarily on affinity-labeling studies dealing with the ribosomal binding site for tRNA and on the interaction of tRNA with mRNA. The results will be correlated with the existing data on the structural organization of the ribosome as obtained by electron and immuno-electron microscopy and used to outline the functional domains on the ribosome for different regions of the tRNA. AFFINITY- AND PHOTOAFFINITY-LABEL DERIVATIVES OF tRNA Position of the Affinity-labeling Group Attachment of an affinity-labeling group to aminoacyl-tRNA requires special precautions to insure single-site specificity. In most studies, advantage has been taken of the existence of the single, highly reactive α -amino group on the aminoacyl moiety of tRNA. Thus, affinity reagents have usually been bound via an amide bond with the...

Yeast tRNA(Asp)-aspartyl-tRNA synthetase complex: low resolution crystal structure.

Yeast aspartyl-tRNA synthetase, a dimer of molecular weight 125,000, and two molecules of its cognate tRNA (Mr = 24160) cocrystallize in the cubic space group I432 (a = 354 A). The crystal structure was solved to low resolution using neutron and X-ray diffraction data. Neutron single crystal diffraction data were collected in five solvents differing by their D2O content in order to use the contrast variation method to distinguish between the protein and tRNA. The synthetase was first located at 40 A resolution using the 65% D2O neutron data (tRNA matched) tRNA molecules were found at 20 A resolution using both neutron and X-ray data. The resulting model was refined against 10 A resolution X-ray data, using density modification and least-squares refinement of the tRNA positions. The crystal structure solved without a priori phase knowledge, was confirmed later by isomorphous replacement. The molecular model of the complex is in good agreement with results obtained in solution by probing the protected part of the tRNA by chemical reagents.

Antisuppressor mutations and sulfur-carrying nucleosides in transfer RNAs of Schizosaccharomyces pombe.

Antisuppressor mutations reduce the efficiency of nonsense suppressors. A mutation in the gene sin4 of Schizosaccharomyces pombe leads to loss of 5-(methoxycarbonylmethyl) thiouridine (mcm5s2U) from the first anticodon position of tRNAs. This resembles the phenotype of sin3 (Heyer, W. D., Thuriaux, P., Kohli, J., Ebert, P., Kersten, H., Gehrke, C., Kuo, K. C., and Agris, P. F. (1984) J. Biol. Chem. 259, 2856-2862), but the mutations reside in different genes. In vivo 35S-labeled tRNA from the parental suppressor strain sup3, the antisuppressor strains sin3 and sin4, and the double mutant sin3 sin4 has been digested to nucleosides and analyzed with high performance liquid chromatography methods. The major sulfur-carrying nucleoside in wild-type S. pombe tRNA is mcm5s2U. It is reduced in the mutant strains. Two other thiolated nucleosides are also present: 2-thiouridine and a nucleoside of unknown structure. Neither was affected by the antisuppressor mutations. Thiocytidine has not been found. Independent from their effect on suppressors, the two mutations sin3 and sin4 reduce the growth rate of cells, and sin3 also increases cell length. In vivo decoding of the serine codon UCG by the UCA reading serine tRNA is not promoted by the two antisuppressor mutations.

Interaction of tRNAPhe and tRNAVal with aminoacyl-tRNA synthetases. A chemical modification study.

The alkylation by ethylnitrosourea of phosphodiester bonds in tRNAPhe from yeast and in tRNAVal from yeast and from rabbit liver and that by 4-(N-2-chloroethyl-N-methylamino)-benzylamine of N-7 atoms of guanosine residues in yeast tRNAVal have been used to study the interaction of these tRNAs with aminoacyl-tRNA synthetases. The modifications occurring at low yield were carried out on 3' and/or 5' end-labelled tRNAs either free or in the presence of cognate or non-cognate synthetases. After splitting of the tRNAs at the alkylated positions, the position of the modification sites in the tRNA sequences were detected by acrylamide gel electrophoresis. It was found that the synthetases protect against alkylation certain phosphate or guanosine residues in their cognate tRNAs. Non-cognate synthetases failed to protect efficiently specific positions in tRNA against modification. In yeast tRNAPhe the cognate phenylalanyl-tRNA synthetase protects certain phosphates located in all four stems and in the anticodon and extra-loop of the tRNA. Particularly strong protections occur on phosphate 34 in the anticodon loop and on phosphates 23, 27, 28, 41 and 46 in the D and anticodon stems. In yeast tRNAVal complexed with yeast valyl-tRNA synthetase the protected phosphates are essentially located in the corner between the amino-acid-accepting and D stems, in the D loop, anticodon stem and in the variable region of the tRNA. Three guanosine residues, located in the D stem, and another one in the 3' part of the anticodon stem were also found protected by the synthetase. In mammalian tRNAVal, complexed with the cognate but heterologous yeast valyl-tRNA synthetase, the protected phosphates lie in the anticodon stem, in the extra-loop and in the T psi arm. The location of the protected residues in the structure of three tRNAs suggests some common features in the binding of tRNAs to aminoacyl-tRNA synthetases. These results will be discussed in the light of informations on interaction sites obtained by nuclease digestion and ultraviolet cross-linking methods.

The yeast tRNATyr gene intron is essential for correct modification of its tRNA product.

Precise deletion of the intervening sequence of a yeast tRNATyr ochre suppressor gene (SUP6) significantly reduced its suppressor activity relative to that of the unaltered gene. This is probably the result of the absence of the pseudouridine modification, normally present at the middle anticodon position of mature suppressor tRNA, in tRNA synthesized in vivo from the deleted gene.

Changes in amount of hypo-modified tRNA having guanine in place of queuine during erythroid differentiation of murine erythroleukemia cells.

The amounts of hypo-modified tRNAs having guanine in place of queuine in murine erythroleukemic cells decreased markedly when the cells differentiated into mature erythroid cells. The amounts of these hypo-modified tRNAs can be determined easily by measuring incorporation of labeled guanine into tRNA with Escherichia coli tRNA--guanine transglycosylase. The decrease was detected at on early stage of erythroid differentiation: namely, before any detectable increase in the percentage of cells containing hemoglobin. The amount of guanine-accepting tRNA species was nearly proportional to the percentage of undifferentiated cells in the population, regardless of the type of inducer used. Decrease in the amounts of hypo-modified tRNAs in the cells was effectively blocked by 12-O-tetradecanoylphorbol 13-acetate, which inhibits differentiation of these cells. 8-Azaguanine, which is known to be substrate of tRNA--guanine transglycosylase, was incorporated almost exclusively into the first position of hypo-modified tRNA in murine erythroleukemic cells when they were pulse-labeled in culture with 8-azaguanine, suggesting strongly that tRNA-guanine transglycosylase in the cells is actually involved in incorporation of 8-azaguanine into tRNA in vivo. The amount of 8-azaguanine incorporated into tRNA in differentiated cells was one third of that in undifferentiated cells, the decrease being parallel with that in the amount of guanine-accepting tRNA in these cells. The results suggest that the appearance of hypo-modified tRNAs in the transformed cells was due to lack of substrate for queuosine biosynthesis in tRNA.

Yeast ochre suppressor SUQ5-ol is an altered tRNA Ser UCA.

Ochre suppressor tRNA was partially purified from strains of Saccharomyces cerevisiae containing the serine-inserting class III suppressor SUQ5-ol. RNA sequence analysis of this tRNA indicated that the suppressor is derived from a UCA-decoding tRNA Ser by a G leads to U substitution in the middle position of the anticodon. The suppressor further differs from the wild-type UCA-decoding tRNA Ser in that the mutant anticodon lacks the modified uridine found in the wobble position of the wild-type tRNA and contains instead another modification in or near the anticodon.

Functional role of ribosylthymine in transfer RNA. Preferential utilization of tRNAs containing ribosylthymine instead of uridine at position 54 in protein synthesis of Dictyostelium discoideum.

In some eucaryotes tRNA species occur containing exclusively uridine or ribosylthymine at position 54. In other eucaryotic tRNA species varying ratios of uridine to ribosylthymine have been found. To elucidate whether variation in the degree of U54 methylation in eucaryotic tRNA plays a functional role in protein synthesis, polysomal and overal cellular tRNAs in the lower eucaryote Dictyostelium discoideum were characterized during vegetative growth and the early developmental preaggregation stage.The nucleoside composition of the tRNAs was determined and U54‐containing tRNA species were detected after methylation in vitro with the rT‐forming enzyme from Escherichia coli. The following results were obtained. (a) During vegetative growth, 85% of the tRNAs present on polysomes contain rT at position 54, whereas tRNA species containing U54 accumulate in non‐polysomal cell fractions. (b) Overall cellular and polysomal tRNAs from the preaggregation stage are less modified with respect to rT54 than those from the vegetative growth stage; in addition pseudo‐uridine (Ψ) and 5‐methylcytidine (m5c) are partially lacking at tRNA positions not yet identified. (c) The rT‐containing tRNAs predominate on polysomes also during preaggregation; however, only 65% of the polysomal tRNAs contain ribosylthymine.The results demonstrate that in D. discoideum, the major class tRNAs utilized for protein synthesis includes molecular species in which a U54 residue has been methylated to rT. The ratio of rT54 to U54 in these tRNAs declines at a physiological stage at which the rate of protein synthesis is reduced. Since in vitro an eucaryotic U54‐containing tRNA of this class translates mRNA less efficiently than its rT‐containing counterpart [K. B. Marcu and B. S. Dudock 1976 Nature (Lond.) 261, 159–162], it is assumed that these U54‐containing tRNAs are rate‐limiting in the elongation of peptide chains in eucaryotes.

Three-dimensional structure of hyper-modified nucleoside Q located in the wobbling position of tRNA

The hyper-modified nucleoside Q (queuosine) is exclusively located in the wobbling position of anticodons of tRNATyr tRNAHis, tRNAAsn and tRNAAsp that recognise codons NAUC (ref. 1). Queuosine and its hexose-containing derivatives are widely distributed in microorganisms, animals and plants. We confirm here the chemical structure of queuosine as 7-(3, 4-trans-4, 5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanosine (Fig. 1). The unique structural features of Q are the unusual cyclopentenediol side chain and the 7-deazaguanine ring. Queuosine is one of the most complicated modified nucleosides found thus far.

Studies with a homogeneous enzyme from rabbit erythrocytes catalyzing the insertion of guanine into tRNA.

An enzyme that catalyzes a post-transcriptional modification of tRNA, resulting in replacement of a base from tRNA by guanine, has been purified 2600-fold from rabbit erythrocyte cytosol. The purest preparation migrates as a single protein band on polycrylamide gel electrophoresis and the enzymatic activity co-electrophoreses with this protein. The native enzyme has a molecular weight of 104,000 and is dissociated into two subunits of Mr= 60,000 and 43,000. The Km for guanine is 1.5 x 10(-7) M and for a pure guanine-accepting tRNA is 3.3 x 10(-9) M. The amino acid composition of the pure enzyme has been determined. To our knowledge this is the first study in which the molecular characteristics of a pure enzyme capable of modifying an internal position in tRNA has been reported.

The identification of the tRNA substrates for thesupKtRNA methylase

Purified preparations of the tRNA methylase deficient in supK strains of Salmonella typhimurium transfer methyl groups from S-adenosylmethionine (SAM) to at least two tRNA species, an alanine tRNA and a serine tRNA. The identity of the tRNA substrates for this enzyme was determined by a change in the elution position of the methyl-labeled tRNA from BND-cellulose columns before and after aminoacylation with a specific amino acid followed by derivatization of the free primary amino group with phenoxy- or naphthoxyacetate. The radioactive methyl group enzymatically added to these tRNAs is both acid and base labile and can be hydrolyzed to a volatile product at pHs above 7.5 and also at pH 1. The methylated 3'-nucleotide isolated from digested tRNA is a pyrimidine derivative and chromatographs like a modified uridylic acid. Its identity has not been established, but it is likely that it corresponds to the methyl ester of V, uridin-5-oxyacetic acid.