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PubMed1981

Transfer ribonucleic Acid modification and its relationship to tumorous and nontumorous plant growth.

Detailed analyses of tRNA hydrolysates from four tissue types of Nicotiana tabacum, pith from intact plants, pith growing in culture, habituated tissue in culture, and crown gall tumor tissue in culture, revealed significant qualitative and quantitative differences in the pattern of methylation. Although pith from intact plants and pith growing in culture possessed seven different methylated nucleosides, only two were found in habituated and tumorous tissues in culture. Four of the five compounds accounting for the difference were tentatively identified as methylated guanosines. Evaluation of results in terms of several parameters, including growth rate, the tumorous state, habituation, tissue culture, and potential for differentiation, indicate that the extent of tRNA methylation may be correlated with the potential for differentiation of a particular tissue.

Journal Article↗
PubMed2002

Another cut for lysine tRNA: application of the hyperprocessing reaction reveals another stabilization strategy in metazoan lysine tRNAs.

Recently, we revealed that the cloverleaf structure of some eukaryotic tRNAs is not always stable in vitro, and the denatured structures of these tRNAs are sometimes detected in bacterial RNase P reactions. We have designated the unusual internal cleavage reaction of these tRNAs as hyperprocessing. We have developed this hyperprocessing strategy as a useful tool for examining the stability of the tRNA cloverleaf structure. There are some common features in such unstable, hyperprocessible tRNAs, and the criteria for the hyperprocessing reaction of tRNA are extracted. Metazoan initiator methionine tRNAs and lysine tRNAs commonly fit the criteria, and are predicted to be hyperprocessible. The RNase P reactions of two metazoan lysine tRNAs from Homo sapiens and Caenorhabditis elegans, which fit the criteria, resulted in resistance to the internal cleavage reaction, while one bacterial lysine tRNA from Acholeplasma laidlawii, which also fits the criteria, was internally cleaved by the RNase P. The results showed that the metazoan lysine tRNAs examined are very stable without base modifications even under in vitro conditions. We also examined the 3'-half short construct of the human lysine tRNA, and the results showed that this RNA was internally cleaved by the enzyme. The results indicated that the human lysine tRNA has the ability to be hyperprocessed but is structurally stabilized in spite of lacking base modifications. A comparative study suggested, moreover, that the acceptor-stem bases should take part in the stabilization of metazoan lysine tRNAs. Our data strongly suggest that the cloverleaf shape of other metazoan lysine tRNAs should also be stabilized by means of similar strategies to in the case of human tRNA(Lys3).

Animals↗
PubMed1984

Processing of tRNA in prokaryotes and eukaryotes.

Considerable progress has been made in defining the steps in the conversion of a tRNA precursor to a mature tRNA. These steps, which differ in different systems, include removal of precursor-specific residues from the 5' and 3' termini of the initial transcript, addition of the 3'-C-C-A terminus, splicing of intervening sequences, and modification of nucleotide residues. Despite these advances in defining the "pathways" of tRNA processing, relatively little is known about most of the enzymes actually involved in these processing steps. In this article I describe the sequence of reactions needed to convert the initial tRNA transcript to a functional, mature tRNA, and discuss the specificity and properties of enzymes known to be involved in this process. In addition, I speculate on the expected specificities of other enzymes involved in tRNA processing which have not yet been identified, and on the structural organization of the processing machinery.

Base Sequence↗
PubMed1993

Translation initiation complex formation with 30 S ribosomal particles mutated at conserved positions in the 3'-minor domain of 16 S RNA.

Escherichia coli 30 S ribosomal subunits containing in vitro (phage T7 RNA polymerase-generated) 16 S rRNA, both wild-type and mutant, were examined by toeprinting. These synthetic particles were used to compare the effects of the absence of base modification and of specific nucleotide substitutions in conserved sequence regions of the RNA on the assembly of mRNA, tRNAs and 30 S particles into a translational initiation complex. Initiation factor-3-dependent selection of tRNA(fMet) from a mixture of tRNA(fMet) and tRNA(Phe) occurred with all particles, although 20 times less initiation factor-3 was needed for the synthetic particles, including the mutants. Whereas isolated 30 S particles or those reconstituted with isolated RNA did not distinguish between tRNA(fMet) and tRNA(Phe) for ternary complex formation in the absence of initiation factor-3 (intrinsic selection ability), the synthetic particles preferred tRNA(fMet). The difference between the natural and synthetic particles appears to be due to the absence of certain base modifications, but not m2(6)A, in the synthetic RNA. Synthetic particles containing the mutation U1512C, which converts the universal U.G pair to C.G enhanced both tRNA(fMet) binding and selectivity, although other mutations at that site, namely U1512G, G1523A and U1512C/C1524U, had no such effect. Mutants U1498G and G1401C/C1501G, both located in a highly conserved single-stranded region of the 3'-minor domain, also enhanced tRNA(fMet) selectivity, in this case by reducing complex formation with elongator tRNA. Complex formation between elongator tRNA and the G1401C/C1501G mutant was reduced to almost undetectable levels. The results also indicated that the association rate for initiation complex formation for G1401C/C1501G was considerably lower than for the wild-type sequence. This result had not been detected by standard tRNA-30 S binding assays. Overall, the data suggest that (some of) the 16 S rRNA base modifications as well as the tertiary structure around the decoding site act to desensitize the intrinsic selection ability of the ribosome for tRNA(fMet).

Base Sequence↗
PubMed1985

Inhibition of aminoacyl-tRNA synthetases by the mycotoxin patulin.

The effect of patulin on tRNA aminoacylation has been determined. This mycotoxin inhibits the aminoacylation process by irreversibly inactivating aminoacyl-tRNA synthetases. At neutral and alkaline pH-values, the inactivation occurs mainly by modification of essential thiol groups of the protein, whereas at acidic pH, where the effect is the most pronounced, the modification of other amino acid residues cannot be excluded.

Amino Acyl-tRNA Synthetases↗
PubMed2000

New chromatographic and biochemical strategies for quick preparative isolation of tRNA.

A combination of hydrophobic chromatography on phenyl-Sepharose and reversed phase HPLC was used to purify individual tRNAs with high specific activity. The efficiency of chromatographic separation was enhanced by biochemical manipulations of the tRNA molecule, such as aminoacylation, formylation of the aminoacyl moiety and enzymatic deacylation. Optimal combinations are presented for three different cases. (i) tRNA(Phe) from Escherichia coli. This species was isolated by a combination of low pressure phenyl-Sepharose hydrophobic chromatography with RP-HPLC. (ii) tRNA(Ile) from E. coli: Aminoacylation increases the retention time for this tRNA in RP-HPLC. The recovered acylated intermediate is deacylated by reversion of the aminoacylation reaction and submitted to a second RP-HPLC run, in which deacylated tRNA(Ile) is recovered with high specific activity. (iii) tRNA(i)(Met) from Saccharomyces cerevisiae. The aminoacylated form of this tRNA is unstable. To increase stability, the aminoacylated form was formylated using E.coli: enzymes and, after one RP-HPLC step, the formylated derivative was deacylated using peptidyl-tRNA hydrolase from E.COLI: The tRNA(i)(Met) recovered after a second RP-HPLC run exhibited electrophoretic homogeneity and high specific activity upon aminoacylation. These combinations of chromatographic separation and biochemical modification can be readily adapted to the large-scale isolation of any particular tRNA.

Acylation↗
PubMed1996

Design, biological activity and NMR-solution structure of a DNA analogue of yeast tRNA(Phe) anticodon domain.

Design of biologically active DNA analogues of the yeast tRNA(Phe) anticodon domain, tDNAPheAC, required the introduction of a d(m5C)-dependent, Mg(2+)-induced structural transition and the d(m1G) disruption of an intra-loop dC.dG base pair. The modifications were introduced at residues corresponding to m5C-40 and wybutosine-37 in tRNA(Phe). Modified tDNAPheAC inhibited translation by 50% at a tDNAPheAC:ribosome ratio of 8:1. The molecule's structure has been determined by NMR spectroscopy and restrained molecular dynamics with an overall r.m.s.d. of 2.8 A and 1.7 A in the stem, and is similar to the tRNA(Phe) anticodon domain in conformation and dimensions. The tDNAPheAC structure may provide a guide for the design of translation inhibitors as potential therapeutic agents.

Anticodon↗
PubMed1997

Biosynthesis of archaeosine, a novel derivative of 7-deazaguanosine specific to archaeal tRNA, proceeds via a pathway involving base replacement on the tRNA polynucleotide chain.

Archaeosine is a novel derivative of 7-deazaguanosine found in transfer RNAs of most organisms exclusively in the archaeal phylogenetic lineage and is present in the D-loop at position 15. We show that this modification is formed by a posttranscriptional base replacement reaction, catalyzed by a new tRNA-guanine transglycosylase (TGT), which has been isolated from Haloferax volcanii and purified nearly to homogeneity. The molecular weight of the enzyme was estimated to be 78 kDa by SDS-gel electrophoresis. The enzyme can insert free 7-cyano-7-deazaguanine (preQ0 base) in vitro at position 15 of an H. volcanii tRNA T7 transcript, replacing the guanine originally located at that position without breakage of the phosphodiester backbone. Since archaeosine base and 7-aminomethyl-7-deazaguanine (preQ1 base) were not incorporated into tRNA by this enzyme, preQ0 base appears to be the actual substrate for the TGT of H. volcanii, a conclusion supported by characterization of preQ0 base in an acid-soluble extract of H. volcanii cells. Thus, this novel TGT in H. volcanii is a key enzyme for the biosynthetic pathway leading to archaeosine in archaeal tRNAs.

Amino Acid Sequence↗
PubMed1997

Intron-dependent enzymatic formation of modified nucleosides in eukaryotic tRNAs: a review.

In eukaryotic cells, especially in yeast, several genes encoding tRNAs contain introns. These are removed from pre-tRNAs during the maturation process by a tRNA-specific splicing machinery that is located within the nucleus at the nuclear envelope. Before and after the intron removal, several nucleoside modifications are added in a stepwise manner, but most of them are introduced prior to intron removal. Some of these early nucleoside modifications are catalyzed by intron-dependent enzymes while most of the others are catalyzed in an intron-independent manner. In the present paper, we review all known cases where the nucleoside modifications were shown to depend strictly on the presence of an intron. These are pseudouridines at anticodon positions 34, 35 and 36 and 5-methylcytosine at position 34 of several eukaryotic tRNAs. One common property of the corresponding intron-dependent modifying enzymes is that their activities are essentially dependent on the local specific architecture of the pre-tRNA molecule that comprises the anticodon stem and loop prolonged by the intron domain. Thus introns clearly serve as internal (cis-type) RNAs that guide nucleoside modifications by providing transient target sites in tRNA for selected nuclear modifying enzymes. This situation may be similar to the recently discovered (trans-type) snoRNA-guided process of ribose methylations of ribosomal RNAs within the nucleolus of eukaryotic cells.

Animals↗
PubMed1995

Codon recognition by tRNA molecules with a modified or unmodified uridine at the first position of the anticodon.

Effects of a single nucleoside modification at the first position of the anticodon of a transfer RNA molecule on its codon reading properties were investigated by use of a cell-free protein synthesis. We prepared two artificial tRNA molecules that differ only in the nucleotide at the first position of the anticodon. One has an unmodified uridine and the other has a 5-methoxyuridine (mo5U). These molecules were charged with labeled serine and introduced into a cell-free protein synthesis directed by a designed mRNA, and the relative codon reading efficiencies were calculated. The results showed that the modification of U into mo5U elevates the reading efficiencies of the UCU and UCG codons but reduces that of the UCA codon.

Anticodon↗
PubMed1997

Posttranscriptional modification of retroviral primers is required for late stages of DNA replication.

During reverse transcription of retroviral RNA, synthesis of (-) strand DNA is primed by a cellular tRNA that anneals to an 18-nt primer binding site within the 5' long terminal repeat. For (+) strand synthesis using a (-) strand DNA template linked to the tRNA primer, only the first 18 nt of tRNA are replicated to regenerate the primer binding site, creating the (+) strand strong stop DNA intermediate and providing a 3' terminus capable of strand transfer and further elongation. On model HIV templates that approximate the (-) strand linked to natural modified or synthetic unmodified tRNA3Lys, we find that a (+) strand strong stop intermediate of the proper length is generated only on templates containing the natural, modified tRNA3Lys, suggesting that a posttranscriptional modification provides the termination signal. In the presence of a recipient template, synthesis after strand transfer occurs only from intermediates generated from templates containing modified tRNA3Lys. Reverse transcriptase from Moloney murine leukemia virus and avian myoblastosis virus shows the same requirement for a modified tRNA3Lys template. Because all retroviral tRNA primers contain the same 1-methyl-A58 modification, our results suggest that 1-methyl-A58 is generally required for termination of replication 18 nt into the tRNA sequence, generating the (+) strand intermediate, strand transfer, and subsequent synthesis of the entire (+) strand. The possibility that the host methyl transferase responsible for methylating A58 may provide a target for HIV chemotherapy is discussed.

DNA Replication↗
PubMed1992

HSD restriction-modification proteins partake in latent anticodon nuclease.

Phage T4-induced anticodon nuclease triggers cleavage-ligation of the host tRNA(Lys). The enzyme is encoded in latent form by the optional Escherichia coli locus prr and is activated by the product of the phage stp gene. Anticodon nuclease latency is attributed to the masking of the core function prrC by flanking elements homologous with type I restriction-modification genes (prrA-hsdM and prrD-hsdR). Activation of anticodon nuclease in extracts of uninfected prr+ cells required synthetic Stp, ATP and GTP and appeared to depend on endogenous DNA. Stp could be substituted by a small, heat-stable E. coli factor, hinting that anticodon nuclease may be mobilized in cellular situations other than T4 infection. Hsd antibodies recognized the anticodon nuclease holoenzyme but not the prrC-encoded core. Taken together, these data indicate that Hsd proteins partake in the latent ACNase complex where they mask the core factor PrrC. Presumably, this masking interaction is disrupted by Stp in conjunction with Hsd ligands. The Hsd-PrrC interaction may signify coupling and mutual enhancement of two prokaryotic restriction systems operating at the DNA and tRNA levels.

DNA Restriction-Modification Enzymes↗
PubMed2002

Crystal structure of archaeosine tRNA-guanine transglycosylase.

Archaeosine tRNA-guanine transglycosylase (ArcTGT) catalyzes the exchange of guanine at position 15 in the D-loop of archaeal tRNAs with a free 7-cyano-7-deazaguanine (preQ(0)) base, as the first step in the biosynthesis of an archaea-specific modified base, archaeosine (7-formamidino-7-deazaguanosine). We determined the crystal structures of ArcTGT from Pyrococcus horikoshii at 2.2 A resolution and its complexes with guanine and preQ(0), at 2.3 and 2.5 A resolutions, respectively. The N-terminal catalytic domain folds into an (alpha/beta)(8) barrel with a characteristic zinc-binding site, showing structural similarity with that of the bacterial queuosine TGT (QueTGT), which is involved in queuosine (7-[[(4,5-cis-dihydroxy-2-cyclopenten-1-yl)-amino]methyl]-7-deazaguanosine) biosynthesis and targets the tRNA anticodon. ArcTGT forms a dimer, involving the zinc-binding site and the ArcTGT-specific C-terminal domain. The C-terminal domains have novel folds, including an OB fold-like "PUA domain", whose sequence is widely conserved in eukaryotic and archaeal RNA modification enzymes. Therefore, the C-terminal domains may be involved in tRNA recognition. In the free-form structure of ArcTGT, an alpha-helix located at the rim of the (alpha/beta)(8) barrel structure is completely disordered, while it is ordered in the guanine-bound and preQ(0)-bound forms. Structural comparison of the ArcTGT.preQ(0), ArcTGT.guanine, and QueTGT.preQ(1) complexes provides novel insights into the substrate recognition mechanisms of ArcTGT.

Amino Acid Sequence↗
PubMed1978

Tumour-specific phenylalanine tRNA contains two supernumerary methylated bases.

Every malignant tumour examined contains aberrant tRNA methyltransferases and a few tRNAs which are absent from the normal tissue of origin. To determine whether tumour-specific tRNAs have different modifications from those in normal tissue, we purified the most frequently occurring tumour-specific isoaccepting tRNA from two malignant tissues. The isoaccepting phenylalanine tRNA from Novikoff hepatoma and Ehrlich ascites cells both contain two supernumerary methylated bases. One of these l-methylguanine, is absent from the phenylalanine tRNA of normal rat, mouse, rabbit and calf liver. An increase in the levels of 5-methylcytidine and dihydrouridine was also detected.

Animals↗
PubMed1998

Efficient ex vivo inhibition of perforin and Fas ligand expression by chimeric tRNA-hammerhead ribozymes.

Graft-versus-host disease (GVHD) is a feared complication of allogeneic bone marrow transplantation. Research in rodent models has linked perforin and Fas ligand (FasL), two components of independent lytic pathways, with the induction of GVHD. In this study we characterized two hammerhead ribozymes that cleave their target perforin and Fas ligand RNAs with high efficiency in CTLL-2 cells. The perforin and Fas ligand ribozymes were expressed from a tRNA-directed RNA polymerase III promoter that was inserted in an episomal multicopy plasmid derived from papilloma virus. Chimeric anti-perforin and anti-FasL tRNA-ribozymes had sequences engineered in order to have specific secondary structure effects. These sequence modifications allow the formation of a 5' --> 3' stem structure and also place the ribozyme in a flexible bulge region that keeps the ribozyme separated from the tRNA domain. Northern and RT in situ PCR analyses showed high levels of transcription and efficient transportation to the cytoplasm. The expression of perforin and FasL in CTLL-2 cells was significantly reduced as assessed by RNA and protein analyses.

Animals↗
PubMed2005

Ribosome-catalyzed synthesis of protein/oligopeptides with unnatural backbone.

Nonsense suppression method was used to probe the allowable modification of substrate (amino acid) backbone in the prokaryotic ribosomal system. Dihydrofolate reductase (DHFR) with an amber mutation was translated in the RF1-diminished prokaryotic cell free translation system in the presence of chemically-misacylated yeast tRNA(Phe)CUA. The prokaryotic ribosome showed a restricted tolerance to the backbone modification. Although natural-type alpha-amino acid was accepted as a good substrate for the ribosome, incorporation of beta-aminopropionic acid was not detected under our experimental conditions. Interestingly, we found that the homologous beta-hydroxyalkanoic acid with elongated methylene (backbone) chain-length can be a substrate for the ribosome, giving an important implication for the chemical mechanism of the ribosome-catalyzed peptide bond forming reaction.

Base Sequence↗
PubMed2005

Dominant-negative mutant phenotypes and the regulation of translation elongation factor 2 levels in yeast.

The eukaryotic translation elongation factor 2 (eEF2), a member of the G-protein superfamily, catalyzes the post-peptidyl transferase translocation of deacylated tRNA and peptidyl tRNA to the ribosomal E- and P-sites. eEF2 is modified by a unique post-translational modification: the conversion of His699 to diphthamide at the tip of domain IV, the region proposed to mimic the anticodon of tRNA. Structural models indicate a hinge is important for conformational changes in eEF2. Mutations of V488 in the hinge region and H699 in the tip of domain IV produce non-functional mutants that when co-expressed with the wild-type eEF2 result in a dominant-negative growth phenotype in the yeast Saccharomyces cerevisiae. This phenotype is linked to reduced levels of the wild-type protein, as total eEF2 levels are unchanged. Changes in the promoter, 5'-untranslated region (5'-UTR) or 3'-UTR of the EFT2 gene encoding eEF2 do not allow overexpression of the protein, showing that eEF2 levels are tightly regulated. The H699K mutant, however, also alters translation phenotypes. The observed regulation suggests that the cell needs an optimum amount of active eEF2 to grow properly. This provides information about a new mechanism by which translation is efficiently maintained.

Gene Expression Regulation, Fungal↗
PubMed1998

An RNA model system for investigation of pseudouridine stabilization of the codon-anticodon interaction in tRNALys, tRNAHis and tRNATyr.

The nucleoside conformation of pseudouridine (psi) was investigated in a series of RNA oligonucleotides and compared with the same sequences containing the parent, unmodified uridine nucleoside. 1H NMR spectroscopy was used to determine the glycosyl conformational preference in pseudouridine systems at the nucleoside level; these experiments were extended to trimers, and ultimately to RNA tetraloop hairpins that are models for the codon-anticodon interaction in tRNA. ROESY 1D and 2D NMR experiments were used to measure the nucleoside conformational preference as a function of temperature. The thermodynamic stability of the RNA tetraloops was also analyzed using UV monitored Tm experiments which established that pseudouridine has a very strong stabilizing effect on double-stranded, base pairing interactions when the modification is located within a base-paired region. This was shown for a tetraloop hairpin model of the codon-anticodon interaction in tRNA(Tyr) which contains a psi at position 35. Pseudouridine also stabilizes double-stranded RNA when the psi modification is in a single-stranded region adjacent to a duplex region as occurs for psi at positions 38 or 39 in tRNA(Lys) and tRNA(His). These results establish that pseudouridine modification of RNA is a powerful and versatile mechanism for stabilizing local RNA structure in both single-stranded and double-stranded regions. Previously postulated roles for pseudouridine as a "conformational switch" are unlikely in light of the increased barrier to rotation about the glycosyl bond upon modification of uridine to pseudouridine. The Tm and NMR data show that local RNA stacking stabilization as a result of psi will stabilize adjacent double-stranded RNA regions such as the codon-anticodon interaction in tRNA.

Anticodon↗