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At least 19 recordsLinked to original sources
PubMed1993

The conformation of tRNA genes. Chemical modification studies.

It has been suggested that eukaryotic tRNA genes might adopt a higher order stem and loop structure to facilitate transcription by interaction of their variably spaced intragenic promoter blocks. Using sodium bisulphite, which reacts specifically with cytosine residues in single-stranded nucleic acids, no deamination of C in the TTCGAA sequence of the 3' ICR of a tRNA(Leu) gene could be detected under conditions which caused 60% deamination of cytosine residues within the loop region of a synthetic cruciform cloned in the same negatively supercoiled plasmid vector. We conclude that, under these conditions, such structures occur in tRNA genes very rarely, if at all.

DNA, Superhelical↗
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↗
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↗
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↗
PubMedDate not supplied

[Tyrosine tRNA(Q*psiA) from bovine liver. Identification of its sites of interaction with homologous aminoacyl-trna synthetase using chemical modification].

Interaction of the bovine liver tyrosine tRNA Q*psi A having short variable loop, with the homologous aminoacyl-tRNA synthetase has been studied by alkylation with ethylnitrosourea. It is shown that tyrosyl-tRNA synthetase (form M.r. 2x39000) protects 3'-phosphates of nucleotides 21 in D-loop, 31 and 44 in anticodon stem and 59-64 T psi C-stem of tRNA Tyr from alkylation. An anticodon of tRNA Tyr did not interact with this proteolytically modified form of tyrosyl-tRNA synthetase (M.r. 2x39000).

Amino Acyl-tRNA Synthetases↗
PubMed2006

The hybrid state of tRNA binding is an authentic translation elongation intermediate.

The GTPase elongation factor (EF)-G is responsible for promoting the translocation of the messenger RNA-transfer RNA complex on the ribosome, thus opening up the A site for the next aminoacyl-tRNA. Chemical modification and cryo-EM studies have indicated that tRNAs can bind the ribosome in an alternative 'hybrid' state after peptidyl transfer and before translocation, though the relevance of this state during translation elongation has been a subject of debate. Here, using pre-steady-state kinetic approaches and mutant analysis, we show that translocation by EF-G is most efficient when tRNAs are bound in a hybrid state, supporting the argument that this state is an authentic intermediate during translation.

Escherichia coli↗
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↗
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↗
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↗
PubMed1987

Comparison of the tertiary structure of yeast tRNA(Asp) and tRNA(Phe) in solution. Chemical modification study of the bases.

A comparative study of the solution structures of yeast tRNA(Asp) and tRNA(Phe) was undertaken with chemical reagents as structural probes. The reactivity of N-7 positions in guanine and adenine residues was assayed with dimethylsulphate and diethyl-pyrocarbonate, respectively, and that of the N-3 position in cytosine residues with dimethylsulphate. Experiments involved statistical modifications of end-labelled tRNAs, followed by splitting at modified positions. The resulting end-labelled oligonucleotides were resolved on polyacrylamide sequencing gels and analysed by autoradiography. Three different experimental conditions were used to follow the progressive denaturation of the two tRNAs. Experiments were done in parallel on tRNA(Asp) and tRNA(Phe) to enable comparison between the two solution structures and to correlate the results with the crystalline conformations of both molecules. Structural differences were detected for G4, G45, G71 and A21: G4 and A21 are reactive in tRNA(Asp) and protected in tRNA(Phe), while G45 and G71 are protected in tRNA(Asp) and reactive in tRNA(Phe). For the N-7 atom of A21, the different reactivity is correlated with the variable variable loop structures in the two tRNAs; in the case of G45 the results are explained by a different stacking of A9 between G45 and residue 46. For G4 and G71, the differential reactivities are linked to a different stacking in both tRNAs. This observation is of general significance for helical stems. If the previous results could be fully explained by the crystal structures, unexpected similarities in solution were found for N-3 alkylation of C56 in the T-loop, which according to crystallography should be reactive in tRNA(Asp). The apparent discrepancy is due to conformational differences between crystalline and solution tRNA(Asp) at the level of the D and T-loop contacts, linked to long-distance effects induced by the quasi-self-complementary anticodon GUC, which favour duplex formation within the crystal, contrarily to solution conditions where the tRNA is essentially in its free state.

Alkylating Agents↗
PubMed1990

[Determination of interacting segments of tRNA(Leu) from cow mammary glands with homologous aminoacyl-tRNA-synthetase by a chemical modification method].

The interaction of the cow mammary gland tRNA(IAGLeu), having a long variable loop, with the cognate aminoacyl-tRNA synthetase has been studied by the alkylation with ethylnitrosourea. It was shown that leucyl-tRNA synthetase protects from alkylation 3'-phosphates of the nucleotides 12-13 in D-loop, 23-24 in D-stem and 37-43 in the anticodon arm of tRNA(IAGLeu). All regions of interaction with the aminoacyl-tRNA synthetase are located in the same plane of tRNA whereas the long variable loop is in another plane.

Alkylation↗
PubMed1998

[Comparative analysis of interaction sites of Thermus thermophilus and Escherichia coli tRNA(Tyr) with homologous aminoacyl-tRNA synthetases by means of chemical modification and nuclease hydrolysis].

A nucleotide sequence of tRNA(Tyr) from the extreme thermophile Thermus thermophilus HB-27 living at 75 degrees C was determined. It is 86 nt long and shares a 52% homology with tRNA(Tyr) from Escherichia coli. A comparative analysis of the interaction sites of tRNA(Tyr) from T. thermophilus and E. coli with the cognate aminoacyl-tRNA synthetases was accomplished by the chemical modification and nuclease hydrolysis approaches. The tRNA(Tyr) was shown to interact with the cognate enzyme in the anticodon stem (on the 5'-side), in the anticodon, in the variable stem and loop (on the 5'-side), and in the acceptor stem (on the 3'-side). These regions are located in the variable stem of the L-form. It was demonstrated that, upon forming the complex E. coli tRNA(Tyr)-cognate synthetase, endonuclease V1 induces additional cleavages of phosphodiester bonds on the 3'-side of the anticodon stem and on the 5'-side of the T-stem. This implies that tRNA may change its conformation when it interacts with the enzyme.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Chemical modification study of aminoacyl-tRNA conformation.

Chemical reactivity of cytosines in 32P-labeled E. coli tRNA1Leu, E. coli tRNAPhe and yeast tRNAPhe before and after aminoacylation was examined by use of a cytosine-specific reagent, semicarbazide-bisulfite mixture. In all the three tRNA species examined, the cytosine residues that were susceptible to the modification were the same in the aminoacylated tRNA and the unacylated tRNA. Only a limited number of the cytosine residues were modifiable: those that occur in the anticodon, the 3'-CCA terminus, the D-loop, and the extra loop. The sites accessible by the reagent are in good agreement with the general three-dimensional structure of tRNA proposed in literature. These results indicate that the gross conformation of these tRNAs does not change on aminoacylation, and consequently favor the view that the T psi C(G) sequence could become exposed in later steps of protein synthesis in order to achieve the binding of aminoacyl tRNA to ribosomes.

Anticodon↗