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PubMed1990

Residual tRNA secondary structure in 'denaturing' 8M urea/TBE polyacrylamide gels: effects on electrophoretic mobility and dependency on prior chemical modification of the tRNA.

Fifteen individual species of tRNA were treated with the chemical modifiers diethylpyrocarbonate, 50% aqueous hydrazine or hydrazine/3 M NaCl. Following purification of the chemically modified material on polyacrylamide gels containing 8 M urea, variant minor bands, in addition to the expected main band, were observed for 12 of the 15 tRNAs. Characterization of the content of chemically altered bases in material recovered from such bands indicated that tRNAs containing modified nucleotides in base-paired stems were excluded from the main band and present, often in enhanced amounts, in the minor variant bands. The persistence of residual secondary structure on 8 M urea gels run at 45 degrees C and the ability of chemically modified bases to alter electrophoretic mobilities warrant caution in designing and interpreting experiments in which chemically modified RNA is isolated on gels prior to further analysis. tRNA(Val) (VAC) was unique in that modified bases in non base-paired regions, according to the cloverleaf model of secondary structure, caused exclusion from the main band. Consequently, we propose a secondary structure for partially denatured tRNA(Val) (VAC), in which these bases are located in double stranded regions of the molecule.

Base Sequence↗
PubMed2004

Identification and characterization of mouse TRMU gene encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase.

The nucleotide modification in tRNA plays a pivotal role in the fidelity of translational process. The mutated mitochondrial tRNA (mt tRNA) associated with human diseases often exhibited a defect in nucleotide modification at wobble position of anticodons. Recently, the product of trmU, 5-methylaminomethyl-2-thiouridylate-methyltransferase, has been shown to be one component of enzyme complex for the biosynthesis of mnm5s2U in the wobble position of the bacterial tRNAs. Here we report the identification and characterization of mouse TRMU homolog. A 1532 bp TRMU cDNA has been isolated and the genomic organization of TRMU has been elucidated. The mouse TRMU gene containing 11 exons encodes a 417 residue protein with a strong homology to the TRMU-like proteins of bacteria and other homologs related to tRNA modification. The mouse TRMU is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver and brain. Furthermore, immunofluorescence analysis of NIH3T3 cells expressing TRMU-GFP fusion protein demonstrated that the mouse Trmu localizes in mitochondria. These observations suggest that the mouse TRMU is a structural and functional homolog of bacterial TrmU, thereby playing a role in the mt tRNA modification and protein synthesis.

3T3 Cells↗
PubMed1996

Crystal structure of tRNA-guanine transglycosylase: RNA modification by base exchange.

tRNA-guanine transglycosylases (TGT) are enzymes involved in the modification of the anticodon of tRNAs specific for Asn, Asp, His and Tyr, leading to the replacement of guanine-34 at the wobble position by the hypermodified base queuine. In prokaryotes TGT catalyzes the exchange of guanine-34 with the queuine (.)precursor 7-aminomethyl-7-deazaguanine (preQ1). The crystal structure of TGT from Zymomonas mobilis was solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 19% at 1.85 angstrom resolution. The structure consists of an irregular (beta/alpha)8-barrel with a tightly attached C-terminal zinc-containing subdomain. The packing of the subdomain against the barrel is mediated by an alpha-helix, located close to the C-terminus, which displaces the eighth helix of the barrel. The structure of TGT in complex with preQ1 suggests a binding mode for tRNA where the phosphate backbone interacts with the zinc subdomain and the U33G34U35 sequence is recognized by the barrel. This model for tRNA binding is consistent with a base exchange mechanism involving a covalent tRNA-enzyme intermediate. This structure is the first example of a (beta/alpha)-barrel protein interacting specifically with a nucleic acid.

Amino Acid Sequence↗
PubMed2006

Rapid tRNA decay can result from lack of nonessential modifications.

The biological role of many nonessential tRNA modifications outside of the anticodon remains elusive despite their evolutionary conservation. We show here that m7G46 methyltransferase Trm8p/Trm82p acts as a hub of synthetic interactions with several tRNA modification enzymes, resulting in temperature-sensitive growth. Analysis of three double mutants indicates reduced levels of tRNA(Val(AAC)), consistent with a role of the corresponding modifications in maintenance of tRNA levels. Detailed examination of a trm8-delta trm4-delta double mutant demonstrates rapid degradation of preexisting tRNA(Val(AAC)) accompanied by its de-aminoacylation. Multiple copies of tRNA(Val(AAC)) suppress the trm8-delta trm4-delta growth defect, directly implicating this tRNA in the phenotype. These results define a rapid tRNA degradation (RTD) pathway that is independent of the TRF4/RRP6-dependent nuclear surveillance pathway. The degradation of an endogenous tRNA species at a rate typical of mRNA decay demonstrates a critical role of nonessential modifications for tRNA stability and cell survival.

Anticodon↗
PubMed2006

tRNA's wobble decoding of the genome: 40 years of modification.

The genetic code is degenerate, in that 20 amino acids are encoded by 61 triplet codes. In 1966, Francis Crick hypothesized that the cell's limited number of tRNAs decoded the genome by recognizing more than one codon. The ambiguity of that recognition resided in the third base-pair, giving rise to the Wobble Hypothesis. Post-transcriptional modifications at tRNA's wobble position 34, especially modifications of uridine 34, enable wobble to occur. The Modified Wobble Hypothesis proposed in 1991 that specific modifications of a tRNA wobble nucleoside shape the anticodon architecture in such a manner that interactions were restricted to the complementary base plus a single wobble pairing for amino acids with twofold degenerate codons. However, chemically different modifications at position 34 would expand the ability of a tRNA to read three or even four of the fourfold degenerate codons. One foundation of Crick's Wobble Hypothesis was that a near-constant geometry of canonical base-pairing be maintained in forming all three base-pairs between the tRNA anticodon and mRNA codon on the ribosome. In accepting an aminoacyl-tRNA, the ribosome requires maintenance of a specific geometry for the anticodon-codon base-pairing. However, it is the post-transcriptional modifications at tRNA wobble position 34 and purine 37, 3'-adjacent to the anticodon, that pre-structure the anticodon domain to ensure the correct codon binding. The modifications create both the architecture and the stability needed for decoding through restraints on anticodon stereochemistry and conformational space, and through selective hydrogen bonding. A physicochemical understanding of modified nucleoside contributions to the tRNA anticodon domain architecture and its decoding of the genome has advanced RNA world evolutionary theory, the principles of RNA chemistry, and the application of this knowledge to the introduction of new amino acids to proteins.

Anticodon↗
PubMedDate not supplied

[Influence of the modification of Phe-tRNA synthetase from Escherichia coli by lysine- and arginine-specific reagent on the ionic interactions of the enzyme with tRNA Phe].

The influence of modification of Phe-RSase from E. coli MRE-600 by lysine- and arginine-specific reagent 2,4-pentandione on the Phe-RSase.tRNAPhe interactions was investigated. It was shown that modification of Phe-RSase with 2,4-pentandion leads to a decrease of the aminoacylation rate without any influence on the value of Km for tRNAPhe in this reaction and only a slight increase of the value of Kdiss for Phe-RSase.tRNAPhe complex. The log Km (Km-1)--ionic strength dependence for native enzyme and log Kdiss (K-1diss) for native enzyme and two forms modified on arginine and lysine residues were investigated. Results were interpreted quantitatively by Debye--Huckel approximation for two spherical macroions and by Daune approximation assuming that the region of tRNA implicated in ionic interactions is locally a cylindrical polyelectrolyte. It was shown that there are 2-4 electrostatic contacts in Phe-RSase.tRNAPhe interactions in limits of both approximations; modification of arginine residues in Phe-RSase doesn't change the number of electrostatic contacts, modification of lysine residues leads to an increase in the number of contacts. It was assumed that there are lysine residues in Phe-RSase essential for the tRNAPhe recognition. The possibility of participation of negative amino acid residues in electrostatic interactions with tRNAPhe is not excluded.

Amino Acyl-tRNA Synthetases↗
PubMed1994

Higher-order structure of bovine mitochondrial tRNA(SerUGA): chemical modification and computer modeling.

On the basis of enzymatic probing and phylogenetic comparison, we have previously proposed that mammalian mitochondrial tRNA(sSer) (anticodon UGA) possess a slightly altered cloverleaf structure in which only one nucleotide exists between the acceptor stem and D stem (usually two nucleotides) and the anticodon stem consists of six base pairs (usually five base pairs) [Yokogawa et al. (1991) Nucleic Acids Res. 19, 6101-6105]. To ascertain whether such tRNA(sSer) can be folded into a normal L-shaped tertiary structure, the higher-order structure of bovine mitochondrial tRNA(SerUGA) was examined by chemical probing using dimethylsulfate and diethylpyrocarbonate, and on the basis of the results a tertiary structure model was obtained by computer modeling. It was found that a one-base-pair elongation in the anticodon stem was compensated for by multiple-base deletions in the D and extra loop regions of the tRNA(SerUGA), which resulted in preservation of an L-shaped tertiary structure similar to that of conventional tRNAs. By summarizing the findings, the general structural requirements of mitochondrial tRNAs necessary for their functioning in the mitochondrial translation system are considered.

Alkylating Agents↗
PubMed1976

Role of modified nucleosides in tRNA: effect of modification of the 2-thiouridine derivative located at the 5'-end of the anticodon of yeast transfer RNA Lys2.

Yeast tRNA Lys2 codes preferentially for AAA and contains a 2-thiouridine derivative (U) at the 5'-position of the anticodon. Removal of the 2-thio group from U by treatment with CNBr did not affect the amino acid accepting activity of the modified tRNA Lys2. CNBr treated tRNA Lys2 was active in protein synthesis but with a much reduced efficiency. Although the modified tRNA Lys2 was recognized by elongation factor (EF) T, the EFT dependent binding to ribosomes to tRNA Lys2 (CNBr) was markedly decreased.

Anticodon↗
PubMed1987

A cell-free plant extract for accurate pre-tRNA processing, splicing and modification.

An intron-containing tobacco tRNA(Tyr) precursor synthesized in a HeLa cell nuclear extract has been used to develop a cell-free processing and splicing system from wheat germ. Removal of 5' and 3' flanking sequences, accurate excision of the intervening sequence, ligation of the resulting tRNA halves, addition of the 3'-terminal CCA sequence and modification of seven nucleosides were achieved in appropriate wheat germ S23 and S100 extracts. The maturation of pre-tRNA(Tyr) in these extracts resembles the pathway observed in vivo for tRNA biosynthesis in Xenopus oocytes and yeast in that processing of the flanks precedes intron excision. Most of the modified nucleosides (m2(2) G, psi 35, psi 55, m7G and m1A) are introduced into the intron-containing pre-tRNA with mature ends, whereas two others (m1G and psi 39) are only found in the mature tRNA(Tyr). Processing and splicing proceed very efficiently in the wheat germ extracts, leading to complete maturation of 5' and 3' ends followed by about 65% conversion to mature tRNA(Tyr) under our standard conditions. The activity of the wheat germ endonuclease is stimulated 3-fold by the non-ionic detergent Triton X-100. All previous attempts to demonstrate the presence of a splicing endonuclease in wheat germ had failed (Gegenheimer et al., 1983). Hence, this is the first cell-free plant extract which supports pre-tRNA processing and splicing in vitro.

Cell Nucleus↗
PubMed2001

Wobble modification defect in tRNA disturbs codon-anticodon interaction in a mitochondrial disease.

We previously showed that in mitochondrial tRNA(Lys) with an A8344G mutation responsible for myoclonus epilepsy associated with ragged-red fibers (MERRF), a subgroup of mitochondrial encephalomyopathic diseases, the normally modified wobble base (a 2-thiouridine derivative) remains unmodified. Since wobble base modifications are essential for translational efficiency and accuracy, we used mitochondrial components to estimate the translational activity in vitro of purified tRNA(Lys) carrying the mutation and found no mistranslation of non-cognate codons by the mutant tRNA, but almost complete loss of translational activity for cognate codons. This defective translation was not explained by a decline in aminoacylation or lowered affinity toward elongation factor Tu. However, when direct interaction of the codon with the mutant tRNA(Lys) defective anticodon was examined by ribosomal binding analysis, the wild-type but not the mutant tRNA(Lys) bound to an mRNA- ribosome complex. We therefore concluded that the anticodon base modification defect, which is forced by the pathogenic point mutation, disturbs codon- anticodon pairing in the mutant tRNA(Lys), leading to a severe reduction in mitochondrial translation that eventually could result in the onset of MERRF.

Animals↗
PubMed1983

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.

Anticodon↗
PubMed1984

Valyl-tRNA synthetase modification-dependent restriction of bacteriophage T4.

A strain of Escherichia coli, CP 790302, severely restricts the growth of wild-type bacteriophage T4. In broth culture, most infections of single cells are abortive, although a few infected cells exhibit reduced burst sizes. In contrast, bacteriophage T4 mutants impaired in the ability to modify valyl-tRNA synthetase develop normally on this strain. Biochemical evidence indicates that the phage-modified valyl-tRNA synthetase in CP 790302 is different from that previously described. Although the enzyme is able to support normal protein synthesis, a disproportionate amount of phage structural protein (serum blocking power) fails to mature into particles of the appropriate density. The results with host strain CP 790302 are consistent with either a gratuitous inhibition of phage assembly by faulty modification or abrogation of an unknown role that valyl-tRNA synthetase might normally play in viral assembly.

Amino Acyl-tRNA Synthetases↗
PubMedDate not supplied

[Specific modification of phenylalanine:tRNA-ligases of E. coli MRE-600 with N-chlorambucilyl-14c-phenylalanyl-tRNA].

N-Chlorambucilyl-[14C]phenylalanyl-tRNA was used for the affinity modification of phenylalanine : tRNA-ligase from E. coli MRE-600. It has been found that N-chlorambucilyl-[14C]phenylalanyl-tRNA selectively inactivates phenylalanine : tRNA-lagase that results in formation of a covalent bond between the tRNA derivative and the enzyme at pH 5.8, 25 degrees C. The rate fall of the aminoacylation of tRNA with [14C]phenylalanine was observed after the enzyme incubation with N-chlorambucilyl-[14C]phenylalanyl-tRNA at pH 7.5, 25 degrees C. It has been shown that this modification results in a similar rate decrease of tRNA aminoacylation with [14C]phenylalanine, ATP-[32P]pyrophosphate exchange and reaction of the enzymatic deacylation of [14C]phenylalanyl-tRNA. This fact evidences in favour of the possibility of the alkylation to proceed in the proximity of the active centre of the enzyme. The covalent complex obtained seems to be an interesting model for the studies of the mechanisms involved in tRNA aminoacylation as well as for elucidation of the tertiary structure of tRNA bound with the enzyme.

Adenosine Triphosphate↗
PubMed1986

Escherichia coli tyrosyl- and methionyl-tRNA synthetases display sequence similarity at the binding site for the 3'-end of tRNA.

Covalent modification of Escherichia coli tyrosyl-tRNA synthetase (TyrRS) by the 2',3'-dialdehyde derivative of tRNATyr (tRNAox) resulted in a time-dependent inactivation of both ATP-PPi exchange and tRNA aminoacylation activities of the enzyme. In parallel with the inactivation, covalent incorporation of approximately 1 mol of [14C]tRNATyrox/mol of the dimeric synthetase occurred. Intact tRNATyr protected the enzyme against inactivation by the tRNA dialdehyde. Treatment of the TyrRS-[14C]tRNATyr covalent complex with alpha-chymotrypsin produced two labeled peptides (A and B) that were isolated and identified by sequence analysis. Peptides A and B are adjacent and together span residues 227-244 in the primary structure of the enzyme. The three lysine residues in this sequence (lysines-229, -234, and -237) are labeled in a mutually exclusive fashion, with lysine-234 being the most reactive. By analogy with the known three-dimensional structure of the homologous tyrosyl-tRNA synthetase from Bacillus stearothermophilus, these lysines should be part of the C-terminal domain which is presumed to bind the cognate tRNA. Interestingly, the labeled TyrRS structure showed significant similarities to the structure around the lysine residue of E. coli methionyl-tRNA synthetase which is the most reactive toward tRNAMetf(ox) (lysine-335) [Hountondji, C., Blanquet, S., & Lederer, F. (1985) Biochemistry 24, 1175-1180].

Amino Acid Sequence↗
PubMedDate not supplied

[Modification of one tRNA recognition site of phenylalanyl-tRNA synthetase from E. coli MRE-600 with N-chlorambucilyl-phenylalanyl-tRNA].

Affinity labelling of phenylalanyl-tRNA synthetase from E. coli MRE-600 with N-chlorambucilyl-phenylalanyl-tRNA results in a binding of 1 mole of the reagent per 1 mole of the enzyme. Exhaustive alkylation of phenylalanyl-tRNA synthetase completely blocks the aminoacylation and partially inhibits the reaction of ATP--[32P]pyrophosphate exchange. Removal of the tRNA moiety of the reagent by hydrolysis of the ester bond N-chlorambucilyl-phenylalanine and terminal adenosine does not result in a restoration of ATP--[32P]pyrophosphate exchange and aminoacylation activity. The latter result may testify a chemical modification of amino acid residues essential for enzymatic activity. Possibility of blocking one of the two tRNA binding sites is discussed.

Adenosine Triphosphate↗
PubMed1983

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.

Alkylation↗
PubMed2000

Effects of phosphorothioate modifications on precursor tRNA processing by eukaryotic RNase P enzymes.

The cleavage mechanism has been studied for nuclear RNase P from Saccharomyces cerevisiae, Homo sapiens sapiens and Dictyostelium discoideum, representing distantly related branches of the Eukarya. This was accomplished by using precursor tRNAs (ptRNAs) carrying a single Rp or Sp-phosphorothioate modification at the normal RNase P cleavage site (position -1/+1). All three eukaryotic RNase P enzymes cleaved the Sp-diastereomeric ptRNA exclusively one nucleotide upstream (position -2/-1) of the modified canonical cleavage site. Rp-diastereomeric ptRNA was cleaved with low efficiency at the modified -1/+1 site by human RNase P, at both the -2/-1 and -1/+1 site by yeast RNase P, and exclusively at the -2/-1 site by D. discoideum RNase P. The presence of Mn(2+ )and particularly Cd(2+) inhibited the activity of all three enzymes. Nevertheless, a Mn(2+ )rescue of cleavage at the modified -1/+1 site was observed with yeast RNase P and the Rp-diastereomeric ptRNA, consistent with direct metal ion coordination to the (pro)-Rp substituent during catalysis as observed for bacterial RNase P enzymes. In summary, our results have revealed common active-site constraints for eukaryotic and bacterial RNase P enzymes. In all cases, an Rp as well as an Sp-phosphorothioate modification at the RNase P cleavage site strongly interfered with the catalytic process, whereas substantial functional interference is essentially restricted to one of the two diastereomers in other RNA and protein-catalyzed hydrolysis reactions, such as those catalyzed by the Tetrahymena ribozyme and nuclease P1.

Animals↗
PubMed2004

The role of modifications in codon discrimination by tRNA(Lys)UUU.

The natural modification of specific nucleosides in many tRNAs is essential during decoding of mRNA by the ribosome. For example, tRNA(Lys)(UUU) requires the modification N6-threonylcarbamoyladenosine at position 37 (t(6)A37), adjacent and 3' to the anticodon, to bind AAA in the A site of the ribosomal 30S subunit. Moreover, it can only bind both AAA and AAG lysine codons when doubly modified with t(6)A37 and either 5-methylaminomethyluridine or 2-thiouridine at the wobble position (mnm(5)U34 or s(2)U34). Here we report crystal structures of modified tRNA anticodon stem-loops bound to the 30S ribosomal subunit with lysine codons in the A site. These structures allow the rationalization of how modifications in the anticodon loop enable decoding of both lysine codons AAA and AAG.

Anticodon↗