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PubMed2000

The Gcd10p/Gcd14p complex is the essential two-subunit tRNA(1-methyladenosine) methyltransferase of Saccharomyces cerevisiae.

The modified nucleoside 1-methyladenosine (m(1)A) is found at position 58 in the TPsiC loop of many eukaryotic tRNAs. The absence of m(1)A from all tRNAs in Saccharomyces cerevisiae mutants lacking Gcd10p elicits severe defects in processing and stability of initiator methionine tRNA (tRNA(i)(Met)). Gcd10p is found in a complex with Gcd14p, which contains conserved motifs for binding S-adenosylmethionine (AdoMet). These facts, plus our demonstration that gcd14Delta cells lacked m(1)A, strongly suggested that Gcd10p/Gcd14p complex is the yeast tRNA(m(1)A)methyltransferase [(m(1)A)MTase]. Supporting this prediction, affinity-purified Gcd10p/Gcd14p complexes used AdoMet as a methyl donor to synthesize m(1)A in either total tRNA or purified tRNA(i)(Met) lacking only this modification. Kinetic analysis of the purified complex revealed K(M) values for AdoMet or tRNA(i)(Met) of 5.0 microM and 2.5 nM, respectively. Mutations in the predicted AdoMet-binding domain destroyed GCD14 function in vivo and (m(1)A)MTase activity in vitro. Purified Flag-tagged Gcd14p alone had no enzymatic activity and was severely impaired for tRNA-binding compared with the wild-type complex, suggesting that Gcd10p is required for tight binding of the tRNA substrate. Our results provide a demonstration of a two-component tRNA MTase and suggest that binding of AdoMet and tRNA substrates depends on different subunits of the complex.

Binding Sites↗

PubMed1988

Purification and characterization of arginyl-tRNA-protein transferase from rabbit reticulocytes. Its involvement in post-translational modification and degradation of acidic NH2 termini substrates of the ubiquitin pathway.

Conjugation of ubiquitin to certain proteins can trigger their degradation. A major question concerns the structural features of a protein which make it susceptible to ubiquitin ligation. Recent studies have shown that the selection of proteins for degradation occurs most probably on a binding site of the ubiquitin-protein ligase (E3). It was shown that a free alpha-NH2 group is one important feature of the protein structure recognized by the ubiquitin-ligating enzyme. Proteins with basic or bulky hydrophobic residues in the NH2-terminal position are recognized by the ligase, marked by ubiquitin, and degraded. This is not true, however, for proteins with an acidic residue in this position. We have previously shown that a tRNA-dependent post-translational conjugation of arginine to acidic NH2 termini of proteins is essential for their degradation via the ubiquitin pathway, and we speculated that this modification is required for their recognition by the ligase. In the present study we have partially purified from rabbit reticulocytes the modifying enzyme, arginyl-tRNA-protein transferase, and characterized it. We have separated the enzyme from other known components of the ubiquitin system and shown that it is specifically required for degradation of proteins with either an aspartate or glutamate residue in their NH2-terminal position. We have shown that the action of the transferase is required for conjugation of ubiquitin to the substrate and most probably for its recognition by the ligase. The enzyme in its native form has a molecular mass of about 360 kDa. It appears to be a complex between several molecules of arginyl-tRNA synthetase and arginyl-tRNA-protein transferase.

Acyltransferases↗

PubMed1985

5-Methylcytidylic modification of in vitro transcript from the rat identifier sequence; evidence that the transcript forms a tRNA-like structure.

Previous studies showed that the rat identifier sequence, the rodent type 2 Alu family, the rabbit C family and the bovine or goat 73 bp repeat show remarkable resemblance with a few specific tRNA molecules (Sakamoto and Okada, J. Mol. Evol. in press). This paper reports 5-methylcytidylic modification of the in vitro transcript from the ID sequence, and provides further evidence that the transcript forms a tRNA-like structure in vitro. Fingerprint analysis and oligonucleotide mapping suggested that the sequence of the oligonucleotide containing 5-methylcytidine is CpCpm5CpUpGp, which corresponds to the extra and T psi stem regions in the secondary structure of phenylalanine tRNA. Since the sequence of the corresponding oligonucleotide in the phenylalanine tRNA is Cpm5CpCpUpGp, these results suggests that a tRNA (cytosine-5) methyl transferase recognizes the secondary structure of the transcript from the ID sequence rather than the primary sequence. The significance of this modification in relation to the functional role of the ID sequence is discussed.

Animals↗

PubMed2002

Wobble modification defect suppresses translational activity of tRNAs with MERRF and MELAS mutations.

By purifying mutant mitochondrial tRNAs, we were able to ascertain that post-transcriptional modification at the anticodon wobble uridine is absent in tRNA(Lys) with the 8344 MERRF mutation and in tRNA(Leu(UUR)) with either the 3243 or 3271 MELAS mutation. Both the MERRF and MELAS mutant tRNAs substantially lost their translational ability, the extent of the loss in each mutant corresponding to the reduction in actual mitochondrial translational activity. Lack of the wobble modification deprived mutant tRNA(Lys) of interaction with the cognate codons. These features indicate that the modification defect plays a primary role in the molecular pathophysiology of these mitochondrial diseases.

Journal Article↗

PubMed2001

Rewiring the keyboard: evolvability of the genetic code.

The genetic code evolved in two distinct phases. First, the 'canonical' code emerged before the last universal ancestor; subsequently, this code diverged in numerous nuclear and organelle lineages. Here, we examine the distribution and causes of these secondary deviations from the canonical genetic code. The majority of non-standard codes arise from alterations in the tRNA, with most occurring by post-transcriptional modifications, such as base modification or RNA editing, rather than by substitutions within tRNA anticodons.

Animals↗

PubMed1993

A modified uridine in the first position of the anticodon of a minor species of arginine tRNA, the argU gene product, from Escherichia coli.

The argU (dnaY) gene product, a minor tRNA(Arg), from Escherichia coli has the anticodon N*CU with an unidentified modified nucleoside N* in position 34 [Kiesewetter, S., Fisher, W. & Sprinzl, M. (1987) Nucleic Acids Res. 15, 3184]. In the present study, argU tRNA was purified from E. coli A19 strain and nucleoside N* was characterized by the TLC and HPLC analyses. Nucleoside N* was found to be different from any naturally occurring modified nucleosides. From unfractionated E. coli tRNA species, nucleoside N* was prepared in an amount sufficient for 1H-NMR experiments. By the analyses of one-dimensional and two-dimensional NMR spectra, nucleoside N* was suggested to be 5-methylaminomethyluridine (mnm5U), which was confirmed by comparison with a chemically synthesized preparation of mnm5U. Thus, the occurrence of mnm5U in mature tRNA was found for the first time. Further, the modification of U(34) to mnm5U in this tRNA was found to contribute to the strict recognition of two degenerate codons terminating in A and G.

Anticodon↗

PubMed1970

Transfer ribonucleic acid in KB cells infected with adenovirus type 2.

Populations of transfer ribonucleic acid (tRNA) extracted from control and type 2 adenovirus (Ad2)-infected KB cells were compared. No consistent differences in acceptor activity for 11 amino acids were observed. Comparison of methylated albumin-kieselguhr (MAK) elution profiles of arginyl-tRNA from control and infected cells revealed a minor modification in that the proportion of arginyl-tRNA eluting at high salt concentration was somewhat greater in infected cells. No similar differences were observed in MAK elution profiles of aspartyl-, isoleucyl-, leucyl-, phenylalanyl-, seryl-, tyrosyl-, and valyl-tRNA. Hybridization of 4S RNA from infected cells labeled by incorporation of (3)H-uridine with Ad2 deoxyribonucleic acid revealed the presence of a complementary species of RNA in this preparation. Hybridization of (3)H-arginyl-tRNA and of (3)H-aminoacyl-tRNA labeled by charging with (3)H-arginine or a (3)H-mixture of amino acids, respectively, failed to detect the presence of virus-specific tRNA in Ad2-infected cells.

Adenoviridae↗

PubMed1979

Purification of Leucine tRNA Isoaccepting Species from Soybean Cotyledons: I. Benzoylated Diethylamino Cellulose Fractionation, N-Hydroxysuccinimide Modification, and Characterization of Product.

Transfer RNA from soybean (Glycine max) cotyledons was purified to homogeneity followed by the purification of the family of leucine tRNA via benzoylated diethylaminoethyl cellulose (BDC) chromatography. Nonacylated total purified tRNA was salicylhydroxamate (SHAM) modified by the phenoxyacetyl method and fractionated into three peaks on a BDC column. The first peak containing bulk tRNA with no hydrophobic character amounted to 78% of the added tRNA. The second peak containing 19% of the added tRNA and represents the tRNA with intrinsic hydrophobic properties. The third peak containing 3% of the tRNA represents the SHAM modified tRNA and nonspecifically modified tRNA. Transfer RNA peaks I and II were pooled and subsequently stoichiometrically acylated in two batches, one containing [(14)C]leucine while the other contained unlabeled leucine. The acylated tRNA was loaded on and step-eluted from a BDC column. The purified acylated-tRNA was phenoxyacetyl modified and following ethanol precipitation was fractionated on a BDC column. A double peak eluted from the column in the ethanol gradient contained 5.3% of the starting optical density and 85.3% of the starting counts per minute. Characterization of this leucine tRNA showed typical ultraviolet spectra properties and appeared to be homogeneous on a G-100 Sephadex column. The minimum purity of the tRNA was 32 to 35%. Finally, the acylated tRNA was chromatographed on an RPC-2 column giving six leucine isoaccepting tRNAs. The data indicate that leucine tRNA was highly purified without losing the integrity of the family of isoacceptors.

Journal Article↗

PubMed2026

Involvement of tRNA thiolation in uORF-mediated translational regulation during Xylogenesis in Arabidopsis thaliana.

Post-transcriptional modification of tRNAs is an important mechanism for regulating translation efficiency and cellular homeostasis, yet its contribution to upstream open reading frame (uORF)-mediated translational control remains largely unexplored. In this study, we investigated the role of tRNA thiolation in thermospermine-dependent regulation of xylem development in Arabidopsis thaliana. Using a suppressor screen of the thermospermine-deficient mutant acaulis5 (acl5), which exhibits dwarfism and excessive xylem differentiation, we identified suppressor-of-acl502 (sac502) as a recessive loss-of-function allele of CTU2, a gene encoding a key enzyme in the biosynthesis of the wobble uridine modification 5-methoxycarbonylmethyl-2-thiouridine. Mutations in other components of the same modification pathway, including ROL5 and TRM9, similarly suppressed the acl5 phenotype. Translational analyses using 5' leader-GUS reporter constructs revealed that the ctu2 mutation did not enhance translation of the mRNA containing a thermospermine-responsive uORF of SAC51, but instead significantly reduced translation of that of SACL3, a member of the SAC51 family, and that of LONESOME HIGHWAY (LHW), which contains another conserved uORF in the 5' leader region. Polysome profiling further demonstrated decreased association of SACL3 and LHW mRNAs with actively translating ribosomes in ctu2. Genetic interaction analyses supported the conclusion that the suppression of excessive xylem formation in acl5 by ctu2 is attributable to reduced LHW activity. In addition, ctu2 mutants displayed increased sensitivity to exogenous thermospermine, resembling the response of lhw mutants. Together, our results reveal that tRNA thiolation contributes to uORF-mediated translational regulation of key developmental regulators and identify tRNA modification as an important regulatory layer controlling vascular development.

Arabidopsis↗

PubMed2002

Highly conserved modified nucleosides influence Mg2+-dependent tRNA folding.

Transfer RNA structure involves complex folding interactions of the TPsiC domain with the D domain. However, the role of the highly conserved nucleoside modifications in the TPsiC domain, rT54, Psi55 and m5C49, in tertiary folding is not understood. To determine whether these modified nucleosides have a role in tRNA folding, the association of variously modified yeast tRNA(Phe) T-half molecules (nucleosides 40-72) with the corresponding unmodified D-half molecule (nucleosides 1-30) was detected and quantified using a native polyacrylamide gel mobility shift assay. Mg2+ was required for formation and maintenance of all complexes. The modified T-half folding interactions with the D-half resulted in K(d)s (rT54 = 6 +/- 2, m5C49 = 11 +/- 2, Psi55 = 14 +/- 5, and rT54,Psi55 = 11 +/- 3 microM) significantly lower than that of the unmodified T-half (40 +/- 10 microM). However, the global folds of the unmodified and modified complexes were comparable to each other and to that of an unmodified yeast tRNA(Phe) and native yeast tRNA(Phe), as determined by lead cleavage patterns at U17 and nucleoside substitutions disrupting the Levitt base pair. Thus, conserved modifications of tRNA's TPsiC domain enhanced the affinity between the two half-molecules without altering the global conformation indicating an enhanced stability to the complex and/or an altered folding pathway.

Base Sequence↗

PubMed1993

A 2-thiouridine derivative in tRNAGlu is a positive determinant for aminoacylation by Escherichia coli glutamyl-tRNA synthetase.

Early investigations into the interaction between Escherichia coli glutamyl-tRNA synthetase (GluRS) and tRNAGlu have implicated the modified nucleoside 5-[(methylamino)methyl]-2-thiouridine in the first position of the anticodon as an important contact for efficient aminoacylation. However, the experimental methods employed were not sufficient to determine whether the interaction was dependent on the presence of the modification or simply involved other anticodon loop-nucleotides, now occluded from interaction with the synthetase. Unmodified E. coli tRNA(Glu), derived by in vitro transcription of the corresponding gene, is a poor substrate for GluRS, exhibiting a 100-fold reduction in its specificity constant (kcat/KM) compared to that of tRNA(Glu) prepared from an overproducing strain. Through the use of recombinant RNA technology, we created several hybrid tRNAs which combined sequences from the in vitro transcript with that of the native tRNA, resulting in tRNA molecules differing in modified base content. By in vitro aminoacylation of these hybrid tRNA molecules and of tRNAs with base substitutions at positions of nucleotide modification, we show conclusively that the modified uridine at position 34 in tRNA(Glu) is required for efficient aminoacylation by E. coli GluRS. This is only the second example of a tRNA modification acting as a positive determinant for interaction with its cognate aminoacyl-tRNA synthetase.

Base Sequence↗

PubMed1989

Binding of the 3' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.

A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF-G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3' end of tRNAPhe, which considerably decrease the affinity of E-site binding, lower the translocation rate up to 40-fold. Furthermore, 3'-end modifications lower or abolish the stimulation by P site-bound tRNA of the GTPase activity of EF-G on the ribosome. The results suggest that a hydrogen-bonding interaction of the 3'-terminal adenine of the leaving tRNA in the E site, most likely base-pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF-G on the ribosome. We propose the following molecular model of translocation: after the binding of EF-G.GTP, the P site-bound tRNA, by a movement of the 3'-terminal single-stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co-operative interaction between the E site and the EF-G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA.

Base Sequence↗

PubMed1976

Amino-terminal arginylation of chromosomal proteins by arginyl-tRNA.

Arginine was transferred from arginyl-tRNA to the amino-terminal end of chromatin proteins by L-arginyl-transferase. The reaction was dependent on the presence of potassium ion and beta-mercaptoethanol and was sensitive to RNase and trypsin. Treatment with DNase partially inhibited the transfer of arginine from arginyl-tRNA suggesting that intact chromatin structure is necessary for modification of chromatin. The radioactivity incorporated into chromatin was sensitive to trypsin but not to DNase or RNase. Most of the incorporated radioactivity was recovered in the phenol fraction, supporting the notion that modification of chromatin takes place in proteins but not in nucleic acids of chromatin. Modification of the proteins by transfer of arginine from arginyl-tRNA takes place mainly in the nonhistone fraction of chromatin. Major portions of chromosomal proteins modified in this manner appear to be released from chromatin. Incubation of incorporated radioactive product with [12C]arginyl-tRNA did not alter the product, showing that incorporated arginine is stable and does not exchange with added arginine or arginyl-tRNA. These observations suggest that aminoacyl-transferase may function in the modification of chromosomal proteins and that modification of chromatin may alter the regulatory mechanisms of cellular functions.

Animals↗

PubMed1981

Total synthesis of a RNA molecule with sequence identical to that of Escherichia coli formylmethionine tRNA.

A RNA molecule has been synthesized that is identical in sequence to Escherichia coli tRNAfMet except that it lacks the base modifications present in the E. coli tRNA. This was achieved by enzymatic joining of chemically synthesized oligonucleotides with chain lengths of 3-10 which were synthesized by the phosphodiester or phosphotriester method. First, quarter molecules of tRNA were constructed by joining of chemically synthesized fragments with RNA ligase. The 5'-quarter molecule (bases 1-20) served as an acceptor in joining reactions with the 3',5'-bisphosphorylated donor molecule (bases 21-34). The 5'-half molecule thus obtained was treated with phosphatase and joined to the 3'-half molecule which was prepared by ligation of the other quarter molecules (bases 35-60, acceptor; bases 61-77, donor) followed by 5'-phosphorylation with polynucleotide kinase. The synthetic tRNA was characterized by oligonucleotide pattern and was partially active in aminoacylation with E. coli methionyl-tRNA synthetase.

Base Sequence↗

PubMed1988

Properties of a transfer RNA lacking modified nucleosides.

A transfer RNA complete devoid of modified nucleosides was synthesized by in vitro transcription, and some of its properties in aminoacylation and protein synthesis in vitro were studied. For this purpose, a plasmid was constructed which contained a glycine tRNA gene from Mycoplasma mycoides under the promoter of the T7 RNA polymerase, as well as a BstNI restriction site at the 3'-end of the tRNA gene. Cleavage of plasmid DNA with BstNI followed by T7 RNA polymerase transcription in vitro yielded an RNA which was processed with M1 RNA, the catalytic subunit of ribonuclease P, to give a tRNA of mature length. The tRNA synthesized in this manner can be esterified with glycine in vitro, and the rate of aminoacylation is the same as when using the corresponding fully modified glycine tRNA from M. mycoides. Furthermore, in protein synthesis in vitro, the tRNA lacking modified nucleosides was essentially as efficient as the corresponding normal glycine tRNA. However, the Escherichia coli extract used in our protein-synthesizing system introduced one modification, pseudouridine, into the in vitro-synthesized tRNA, and it cannot be excluded that this modification has an essential role in protein synthesis.

Adenosine↗

PubMed2000

Modified constructs of the tRNA TPsiC domain to probe substrate conformational requirements of m(1)A(58) and m(5)U(54) tRNA methyltransferases.

The TPsiC stem and loop (TSL) of tRNA contains highly conserved nucleoside modifications, m(5)C(49), T(54), Psi(55)and m(1)A(58). U(54)is methylated to m(5)U (T) by m(5)U(54)methyltransferase (RUMT); A(58)is methylated to m(1)A by m(1)A(58)tRNA methyltransferase (RAMT). RUMT recognizes and methylates a minimal TSL heptadecamer and RAMT has previously been reported to recognize and methylate the 3'-half of the tRNA molecule. We report that RAMT can recognize and methylate a TSL heptadecamer. To better understand the sensitivity of RAMT and RUMT to TSL conformation, we have designed and synthesized variously modified TSL constructs with altered local conformations and stabilities. TSLs were synthesized with natural modifications (T(54)and Psi(55)), naturally occurring modifications at unnatural positions (m(5)C(60)), altered sugar puckers (dU(54)and/or dU(55)) or with disrupted U-turn interactions (m(1)Psi(55)or m(1)m(3)Psi(55)). The unmodified heptadecamer TSL was a substrate of both RAMT and RUMT. The presence of T(54)increased thermal stability of the TSL and dramatically reduced RAMT activity toward the substrate. Local conformation around U(54)was found to be an important determinant for the activities of both RAMT and RUMT.

Animals↗

PubMed1977

Modification of ribonucleic acid by vitamin B6. 1. Specific interaction of pyridoxal 5'-phosphate with transfer ribonucleic acid.

Whole tRNA preparation obtained from a human cell line (HT-29) of colon carcinoma and purified specific Escherichia coli tRNA were reacted with pyridoxal 5'-phosphate, reduced by sodium borohydride and digested with RNase A and snake venom phosphodiesterase. Two-dimensional chromatography of the pyridoxal 5'-phosphate treated tRNA digest showed that pyridoxal 5'-phosphate binds specifically to GMP, presumably in the form of a Schiff base with the exocyclic amino group of the purine. The reaction of pyridoxal 5'-phosphate with whole tRNA was competitively inhibited by N-acetoxy-2-acetylaminofluorene. This suggests that binding occurred primarily to the G20 base residue at the unpaired region of the dihydrouridine loop (Fujimura et al., 1972). The modification of tRNA by pyridoxal 5'-phosphate resulted in the inhibition, to varying extent (10-80%), of amino acid acceptance in the aminoacyl-tRNA synthetase reaction. Defects in codon recognition by pyridoxal 5'-phosphate modified amino acid acylated tRNAs in the presence of the corresponding guanine-containing polynucleotide triplets were observed by the ribosomal binding assay.

Amino Acyl-tRNA Synthetases↗

PubMed1998

Effect of a mutation in the anticodon of human mitochondrial tRNAPro on its post-transcriptional modification pattern.

Although the gene sequences of all 22 tRNAs encoded in the human mitochondrial genome are known, little information exists about their sequences at the RNA level. This becomes a crucial limitation when searching for a molecular understanding of the growing number of maternally inherited human diseases correlated with point mutations in tRNA genes. Here we describe the sequence of human mt-tRNAPropurified from placenta. It shows absence of editing events in this tRNA and highlights the presence of eight post-transcriptional modifications. These include T54, never found so far in an animal mt-tRNA, and m1G37, a modification known to have fundamental functional properties in a number of canonical tRNAs. Occurrence of m1G37 was further investigated in an analysis of the substrate properties of in vitro transcripts of human mt-tRNAProtowards pure Escherichia coli methylguanosine transferase. This enzyme properly methylates G37 in mt-tRNA and is sensitive to the presence of a second G at position 36, neighboring the target nucleotide for methylation. Since mutation of nt 36 was shown to be correlated with myopathy, the potential consequences of non-modification or under-modification of mt-tRNA nucleotides in expression of the particular myopathy and of mitochondrial diseases in general are discussed.

Anticodon↗