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PubMed1970

Structure and function of E. coli formylmethionyl tRNA. I. Effect of modification of pyrimidine residues on aminoacyl synthetase recognition.

E. coli formylmethionyl tRNA (tRNA(fMet)) has been irradiated with ultraviolet light in the presence of Mg(2+) to the extent of 50 per cent inactivation of amino acid acceptance. Separation of active and inactive molecules after irradiation has shown that ultraviolet light modification of the uridine in the anticodon, the uridine in the small loop, the 4-thiouridine, and the pyrimidines in the double-stranded stem adjacent to the dihydrouridine loop has no effect on aminoacylation or transformylation. The ultraviolet light-induced inactivation of methionine acceptance by tRNA(fMet) is due almost entirely to modification of the cytidine residues in the 3'-terminal CCA-OH sequence.

Amino Acids↗
PubMed2004

Codon-specific translational defect caused by a wobble modification deficiency in mutant tRNA from a human mitochondrial disease.

Point mutations in the mitochondrial (mt) tRNA(Leu(UUR)) gene are responsible for mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), a subgroup of mitochondrial encephalomyopathic diseases. We previously showed that mt tRNA(Leu(UUR)) with an A3243G or T3271C mutation derived from patients with MELAS are deficient in a normal taurine-containing modification (taum5U; 5-taurinomethyluridine) at the anticodon wobble position. To examine decoding disorder of the mutant tRNA due to the wobble modification deficiency independent of the pathogenic point mutation itself, we used a molecular surgery technique to construct an mt tRNA(Leu(UUR)) molecule lacking the taurine modification but without the pathogenic mutation. This "operated" mt tRNA(Leu(UUR)) without the taurine modification showed severely reduced UUG translation but no decrease in UUA translation. We thus concluded that the UUG codon-specific translational defect of the mutant mt tRNAs(Leu(UUR)) is the primary cause of MELAS at the molecular level. This result could explain the complex I deficiency observed clinically in MELAS.

Base Sequence↗
PubMed1988

Reaction of nucleic acid bases with alpha-acetylenic esters. Part IV. Preparation of an alkylating derivative of tRNA(Phe) by conformation-specific chemical modification.

The reaction of yeast tRNA(Phe) with methyl chlorotetrolate, ClCH2-C identical to C-COOCH3, was studied. This reagent converts adenine and cytosine rings into derivatives in which an additional heterocycle bearing the alkylating chloromethyl group is fused to the original base; these derivatives can exist in two isomeric forms. Modified nucleosides of this type can be easily identified by reverse-phase HPLC. It was found that under native conditions, the modification of tRNA involves the anticodon loop and the 3'-end. The isomers of adenine derivatives formed in the anticodon loop were different from those formed in the 3'-end. It is suggested that the isomeric structure of the derivatives is related to the fine conformational differences between these two regions of tRNA(Phe). Methyl chlorotetrolate could thus be used as a conformational probe of single-stranded nucleic acids. Preliminary assays showed that modified tRNA(Phe) binds irreversibly to yeast phenylalanyl-tRNA synthetase.

Alkylation↗
PubMed1996

Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a single Rp-phosphorothioate modification in the precursor tRNA at the RNase P cleavage site.

To study the cleavage mechanism of bacterial Nase P RNA, we have synthesized precursor tRNA substrates carrying a single Rp- or Sp-phosphorothioate modification at the RNase P cleavage site. Both the Sp- and the Rp-diastereomer reduced the rate of processing by Escherichia coli RNase P RNA at least 1000-fold under conditions where the chemical step is rate-limiting. The Rp-modification had no effect and the Sp-modification had a moderate effect on precursor tRNA ground state binding to RNase P RNA. Processing of the Rp-diastereomeric substrate was largely restored in the presence of the "thiophilic" Cd2+ as the only divalent metal ion, demonstrating direct metal ion coordination to the (pro)-Rp substituent at the cleavage site and arguing against a specific role for Mg(2+)-ions at the pro-Sp oxygen. For the Rp-diastereomeric substrate, Hill plot analysis revealed a cooperative dependence upon [Cd2+] of nH = 1.8, consistent with a two-metal ion mechanism. In the presence of the Sp-modification, neither Mn2+ nor Cd2+ was able to restore detectable cleavage at the canonical site. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Dramatic inhibition of the chemical step by both the Rp- and Sp-phosphorothioate modification is unprecedented among known ribozymes and points to unique features of transition state geometry in the RNase P RNA-catalyzed reaction.

Base Sequence↗
PubMed1975

The structural basis for the resistance of Escherichia coli formylmethionyl transfer ribonucleic acid to cleavage by Escherichia coli peptidyl transfer ribonucleic acid hydrolase.

Escherichia coli formylmethionly-tRNA-tMet is unique among N-acylaminoacyl-tRNAs in its resistance to cleavage by peptidyl-tRNA hydrolase. Chemical modification of tRNA-fMet with sodium bisulfite converts fMet-tRNA-fMet into a good substrate for the hydrolase. The products of the enzymatic cleavage are free tRNA-fMet and formylmethionine. Bisulfite treatment produces cytidine to uridine base changes at several sites in the tRNA structure. One of these modifications results in formation of a new hydrogen-bonded base pair at the end of the acceptor stem of tRNA-fMet. We have shown that this modification is responsible for the observed change in biological activity. Enzymatic cleavage appears to be facilitated by the presence of a 5-terminal phosphate at the end of a fully base-paired acceptor stem, because removal of the 5-phosphate group from N-acetylphenylalanyl-tRNA-Phe or bisulfite-modified fMet-tRNA-FMet reduced the rate of hydrolysis of these substrates. The unpaired base at the 5 terminus of unmodified fMet-tRNA-fMet appears to reduce susceptibility of the tRNA to hydrolytic attack both by positioning the 5-phosphate in an unfavorable orientation and by directly interfering with enzymatic binding. The unusual structure of the acceptor stem of this E. coli tRNA thus plays a critical role in maintaining the viability of the organism by preventing enzymatic cleavage of the fMet group from the bacterial initiator tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed2003

Naturally-occurring modification restricts the anticodon domain conformational space of tRNA(Phe).

Post-transcriptional modifications contribute chemistry and structure to RNAs. Modifications of tRNA at nucleoside 37, 3'-adjacent to the anticodon, are particularly interesting because they facilitate codon recognition and negate translational frame-shifting. To assess if the functional contribution of a position 37-modified nucleoside defines a specific structure or restricts conformational flexibility, structures of the yeast tRNA(Phe) anticodon stem and loop (ASL(Phe)) with naturally occurring modified nucleosides differing only at position 37, ASL(Phe)-(Cm(32),Gm(34),m(5)C(40)), and ASL(Phe)-(Cm(32),Gm(34),m(1)G(37),m(5)C(40)), were determined by NMR spectroscopy and restrained molecular dynamics. The ASL structures had similarly resolved stems (RMSD approximately 0.6A) of five canonical base-pairs in standard A-form RNA. The "NOE walk" was evident on the 5' and 3' sides of the stems of both RNAs, and extended to the adjacent loop nucleosides. The NOESY cross-peaks involving U(33) H2' and characteristic of tRNA's anticodon domain U-turn were present but weak, whereas those involving the U(33) H1' proton were absent from the spectra of both ASLs. However, ASL(Phe)-(Cm(32),Gm(34),m(1)G(37),m(5)C(40)) exhibited the downfield shifted 31P resonance of U(33)pGm(34) indicative of U-turns; ASL(Phe)-(Cm(32),Gm(34),m(5)C(40)) did not. An unusual "backwards" NOE between Gm(34) and A(35) (Gm(34)/H8 to A(35)/H1') was observed in both molecules. The RNAs exhibited a protonated A(+)(38) resulting in the final structures having C(32).A(+)(38) intra-loop base-pairs, with that of ASL(Phe)-(Cm(32),Gm(34),m(1)G(37),m(5)C(40)) being especially well defined. A single family of low-energy structures of ASL(Phe)-(Cm(32),Gm(34), m(1)G(37),m(5)C(40)) (loop RMSD 0.98A) exhibited a significantly restricted conformational space for the anticodon loop in comparison to that of ASL(Phe)-(Cm(32),Gm(34),m(5)C(40)) (loop RMSD 2.58A). In addition, the ASL(Phe)-(Cm(32),Gm(34),m(1)G(37),m(5)C(40)) average structure had a greater degree of similarity to that of the yeast tRNA(Phe) crystal structure. A comparison of the resulting structures indicates that modification of position 37 affects the accuracy of decoding and the maintenance of the mRNA reading frame by restricting anticodon loop conformational space.

Anticodon↗
PubMed2003

Decoding property of C5 uridine modification at the wobble position of tRNA anticodon.

Post-transcriptional modification at the first (wobble) position of the tRNA anticodon participates in precise decoding of the genetic code. We recently identified a novel taurine-containing modified uridine (tau m5U; 5-taurinomethyluridine) at the wobble position of mammalian mitochondrial tRNAs and found lack of this modification in mutant mitochondrial tRNAs from human pathogenic cells of the mitochondrial encephalomyopathies, investigate molecular pathogenesis of the diseases, decoding activity of wobble uridines with or without C5 modification was measured using E. coli cell-free translation system. It has been revealed that C5 modification has a functional role for stabilizing U:G wobble base pair.

Amino Acid Sequence↗
PubMed2025

MapID-based quantitative mapping of chemical modifications and expression of human transfer RNA.

Detection and quantification of tRNA chemical modifications are critical for understanding their regulatory functions in biology and diseases. However, tRNA-seq-based methods for modification mapping encountered challenges both experimentally (poor processivity of heavily modified tRNAs during reverse transcription or RT) and bioinformatically (frequent reads misalignment to highly similar tRNA genes). Here, we report "MapID-tRNA-seq" where we deployed an evolved reverse transcriptase (RT-1306) into tRNA-seq and developed "MapIDs" that reduce redundancy of the human tRNA genome and explicitly annotate genetic variances. RT-1306 generated robust mutations against m1A and m3C, and RT stops against multiple bulky roadblock modifications. MapID-assisted data processing enabled systematic exclusion of false-positive discoveries of modifications which arise from reads misalignment onto similar genes. We applied MapID-tRNA-seq into mapping m1A, m3C and expression levels of tRNAs in three mammary cell lines, which revealed cell-type dependent modification sites and potential translational regulation of the reduced mitochondrial activities in breast cancer.

Humans↗
PubMed2006

A gene involved in modifying transfer RNA is required for fungal pathogenicity and stress tolerance of Colletotrichum lagenarium.

7-Methylguanosine (m7G) modification of tRNA occurs widely in prokaryotes and eukaryotes, although information about its biological roles is limited. Here, we report that a gene involved in m7G modification of tRNA is required for infection by the phytopathogenic fungus Colletotrichum lagenarium. Analysis of the infection-deficient mutant of C. lagenarium, produced by plasmid insertional mutagenesis, identified a tagged gene that is designated APH1. The aph1 mutants, generated by targeted gene disruption, exhibit significant reduction in pathogenicity on the host plants. We conclude that APH1 is required for fungal infection in C. lagenarium. Aph1 showed a strong similarity to Saccharomyces cerevisiae Trm8 involved in m7G modification of tRNA. The m7G content of tRNA from the aph1 deletion mutant was severely reduced compared with that from the wild type, indicating that APH1 is required for m7G methyltransferase activity. Appressoria formed by the aph1 mutants developed penetration hyphae into cellophane, suggesting that appressoria of the mutants retain basic function for penetration. However, the aph1 mutants failed to develop intracellular penetration hyphae into epidermis of the host plants, suggesting a specific requirement of APH1 for appressorium-mediated host invasion. The mutants also had increased sensitivity to salinity and H2O2 stresses. Interestingly, a heat shock treatment on the host plants enabled the aph1 mutant to penetrate them. These data suggest that the APH1 is required for the plant invasion, probably to overcome environmental stresses derived from basal preinvasion (penetration) defence of the host plants.

Colletotrichum↗
PubMed1977

A rapid cytosine-specific modification of E. coli tRNA Leu 1 by semicarbazide-bisulfite, a probe for polynucleotide conformations.

Cytosine residues in 32P-labeled E. coli tRNA Leu 1 were modified by treatment of the tRNA with the semicarbazide-bisulfite reagents [Hayatsu, H. (1976) Biochemistry 15, 2677-2682]. Analysis of the modification sites showed that only four cytidine residues, i.e. C35, C53, C85 and C86, reacted. They were identical with the cytidines of this tRNA accessible to methoxyamine [Chang, S. E. and Ish-Horowicz, D. (1974) J. Mol. Biol. 84, 375-388] and the accessibility was consistent with the conformational features recognized for tRNA in general. The rapidity and the simple nature of this modification demonstrate that the semicarbazide-bisulfite reaction is a useful tool in studying conformations of polynucleotides.

Base Sequence↗
PubMed2000

tRNA-guanine transglycosylase from Escherichia coli: recognition of noncognate-cognate chimeric tRNA and discovery of a novel recognition site within the TpsiC arm of tRNA(Phe).

tRNA-guanine transglycosylase (TGT) is a key enzyme involved in the posttranscriptional modification of tRNA across the three kingdoms of life. In eukaryotes and eubacteria, TGT is involved in the introduction of queuine into the anticodon of the cognate tRNAs. In archaebacteria, TGT is responsible for the introduction of archaeosine into the D-loop of the appropriate tRNAs. The tRNA recognition patterns for the eubacterial (Escherichia coli) TGT have been studied. These studies are all consistent with a restricted recognition motif involving a U-G-U sequence in a seven-base loop at the end of a helix. While attempting to investigate the potential of negative recognition elements in noncognate tRNAs via the use of chimeric tRNAs, we have discovered a second recognition site for the E. coli TGT in the TpsiC arm of in vitro-transcribed yeast tRNA(Phe). Kinetic analyses of synthetic mutant oligoribonucleotides corresponding to the TpsiC arm of the yeast tRNA(Phe) indicate that the specific site of TGT action is G53 (within a U-G-U sequence at the transition of the TpsiC stem into the loop). Posttranscriptional base modifications in tRNA(Phe) block recognition by TGT, most likely due to a stabilization of the tRNA structure such that G53 is inaccessible to TGT. These results demonstrate that TGT can recognize the U-G-U sequence within a structural context that is different than the canonical U-G-U in the anticodon loop of tRNA(Asp). Although it is unclear if this second recognition site is physiologically relevant, this does suggest that other RNA species could serve as substrates for TGT in vivo.

Base Sequence↗
PubMed1994

Structural elements in yeast tRNAs required for homologous modification of guanosine-26 into dimethylguanosine-26 by the yeast Trm1 tRNA-modifying enzyme.

In eukaryotic tRNAs, guanosines in position 26 (G26), located at the junction between the D-stem and the anticodon stem of tRNA, are usually modified to N2,N2-dimethylguanosine (m2(2)G). Although G26 is a prerequisite for biosynthesis of m2(2)G26, it is not self-sufficient for the formation of the dimethylated G26, since in exceptional cases eukaryotic tRNAs have an unmodified G26. In the yeast Saccharomyces cerevisiae the only tRNA species with an unmodified G26 is tRNAAsp. Using in vitro transcripts of this tRNA, as well as of yeast tRNAPhe, a tRNA containing m2(2)G26 in vivo, we have investigated the requirements on tRNA sequences and structures for the formation of m2(2)G26 by the yeast enzyme, i.e. in a homologous in vitro system. We have now demonstrated that G26 was efficiently dimethylated in vitro also after deletion of the entire anticodon stem and loop. We conclude that the elements necessary for a productive interaction between G26 in nuclear coded yeast tRNAs and the yeast G26 modifying enzyme are located within the core of the tRNA. For modification of G26 to m2(2)G26 via monomethylated G26, important primary and secondary structural elements in the tRNAs are a size of at least five nucleotides in the variable loop together with two G-C base pairs in the D-stem. This is the first case reported where the minimal requirements on nuclear coded tRNAs for a yeast modifying enzyme has been elucidated.

Base Sequence↗
PubMed1993

Discrimination between initiation and elongation of protein biosynthesis in yeast: identity assured by a nucleotide modification in the initiator tRNA.

Cytoplasmic initiator tRNAs from plants and fungi possess an unique 2'-phosphoribosyl residue at position 64 of their sequence. In yeast tRNA(iMet), this modified nucleotide located in the T-stem of the tRNA is a 2'-1''-(beta-O-ribofuranosyl-5''-phosphoryl)-adenosine. The phosphoribosyl residue of this modified nucleoside was removed chemically by treatment involving periodate oxidation of tRNA(iMet) and regeneration of the 3'-terminal adenosine with ATP (CTP):tRNA nucleotidyl transferase. The role of phosphoribosylation at position 64 for interaction with elongation factor eEF-1 alpha and initiation factor 2 (eIF-2) was investigated in the homologous yeast system. Whereas the 5'-phosphoribosyl residue prevents the binding of Met-tRNA(iMet) to eEF-1 alpha, it does not influence the interaction with eIF-2. After removal of the ribosyl group, the demodified initiator tRNA showed binding to eEF-1 alpha, but no change was detected with respect to the interaction with the initiation factor eIF-2. This observation is interpreted to mean that a single modification of an eucaryotic initiator tRNA in yeast serves as a negative discriminant for eEF-1 alpha, thus preventing the initiator tRNA(iMet) from entering the elongation cycle of protein biosynthesis.

Chromatography, Affinity↗
PubMedDate not supplied

[The identification of the sites of tRNA(Ser)(GCU) interaction in bovine liver with homologous aminoacyl-tRNA-synthetase by the chemical modification method].

Interaction of the bovine liver tRNA(GCUSer) having a long variable loop, with the cognate aminoacyl-tRNA synthetase has been studied by alkylation with ethylnitrosourea. It was shown that seryl-tRNA synthetase protects 3'-phosphates of nucleotides 12, 13 in D-stem and 45-47-, 47 G.-, 47 H-variable stem of tRNA(GCUreS) from alkylation. An anticodon loop of tRNA(GCUSer) did not interact with seryl-tRNA synthetase.

Alkylation↗
PubMed2024

TGIRT-seq to profile tRNA-derived RNAs and associated RNA modifications.

RNA modifications are key regulators for RNA processes. tRNA-derived RNAs are small RNAs with size between 15 and 50 bases long that are processed from mature or precursor tRNAs. Despite their more recent discovery, tRNA-derived RNAs have been found to play regulatory roles in many cellular processes including gene silencing, protein synthesis, stress response, and transgenerational inheritance. Furthermore, tRNA-derived RNAs are highly abundant in bodily fluids, posing as potential biomarkers. A unique feature of tRNA-derived RNAs is that they are rich in RNA modifications. Many of the RNA modifications on tRNA-derived RNAs disrupt Watson-Crick base pairing and will thus stall reverse transcriptase, such as N1-methyladenosine (m1A), N1-methylguanosine (m1G) and N2, N2-dimethylguanosine (m22G). These RNA modifications add another layer of regulation onto tRNA-derived RNAs' functions and are of interests for future research. However, these RNA modifications could also lead to lower detection of modification-containing RNAs in genome-wide small RNA sequencing analysis due to reverse transcriptase stall. To circumvent this bias, TGIRT (Thermostable Group II Intron Reverse Transcriptase) has been used to readthrough RNA modifications inserting mismatches. These mismatch signatures can then be used to precisely map the modification sites at base resolution. Here we describe the step-by-step experimental protocol to start with purified RNAs from cells or tissues and use TGIRT to make small RNA sequencing library for Illumina sequencing to profile the abundance of tRNA-derived RNAs and the associated RNA modifications.

RNA, Transfer↗
PubMed1994

Nucleoside modifications stabilize Mg2+ binding in Escherichia coli tRNA(Val): an imino proton NMR investigation.

The structures of in vitro transcribed Escherichia coli tRNA(Val), which lacks base modifications, and the native tRNA, which contains them, are very similar in the presence of excess Mg2+ (Kintanar, Yue, and Horowitz, unpublished results). To further probe the effects of base modifications on the structure of tRNA, the Mg2+ ion dependence of the downfield region of the 1H NMR spectrum of in vitro transcribed E. coli tRNA(Val) in aqueous phosphate buffer was investigated. The spectra indicate a remarkable conformational change in unmodified E. coli tRNA(Val) coincident with binding or release of Mg2+. Assignment of the imino proton resonances in the low Mg2+ form of the tRNA transcript allows a detailed description of the conformational change. There is near total disruption of the D stem and tertiary interactions in the absence of bound Mg2+. A new strong interaction between the U67-A6 base pair and the G50-U64 wobble pair is observed, indicating a substantial structural rearrangement at the junction of the acceptor and T stems. The binding constants of the strong Mg2+ binding sites in the D loop and near the D stem in unmodified tRNA(Val) are at least 2 orders of magnitude less than in tRNAVal containing base modifications. The metal ion binding site in the anticodon loop is somewhat stronger than metal ion binding sites in the D loop and stem in unmodified tRNA(Val), but it is still weaker than all strong Mg2+ binding sites in native tRNA(Val). Thus, one role of the base modifications found in tRNA is to stabilize or strengthen the Mg2+ binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2000

Inhibition of selenoprotein synthesis by selenocysteine tRNA[Ser]Sec lacking isopentenyladenosine.

A common posttranscriptional modification of tRNA is the isopentenylation of adenosine at position 37, creating isopentenyladenosine (i(6)A). The role of this modified nucleoside in protein synthesis of higher eukaryotes is not well understood. Selenocysteyl (Sec) tRNA (tRNA([Ser]Sec)) decodes specific UGA codons and contains i(6)A. To address the role of the modified nucleoside in this tRNA, we constructed a site-specific mutation, which eliminates the site of isopentenylation, in the Xenopus tRNA([Ser]Sec) gene. Transfection of the mutant tRNA([Ser]Sec) gene resulted in 80% and 95% reduction in the expression of co-transfected selenoprotein genes encoding type I and II iodothyronine deiodinases, respectively. A similar decrease in type I deiodinase synthesis was observed when transfected cells were treated with lovastatin, an inhibitor of the biosynthesis of the isopentenyl moiety. Neither co-transfection with the mutant tRNA gene nor lovastatin treatment reduced type I deiodinase mRNA levels. Also, mutant tRNA expression did not alter initiation of translation or degradation of the type I deiodinase protein. Furthermore, isopentenylation of tRNA([Ser]Sec) was not required for synthesis of Sec on the tRNA. We conclude that isopentenylation of tRNA([Ser]Sec) is required for efficient translational decoding of UGA and synthesis of selenoproteins.

Animals↗