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PubMed1972

Different arginine transfer ribonucleic acid species prevalent in shaken and unshaken cultures of Neurospora.

When the arginyl-transfer ribonucleic acid (tRNA) species isolated from unshaken and from shaken cultures of Neurospora were compared by co-chromatography, a marked change in the relative abundance of the two main tRNA(arg) species was found. The two arginine tRNA species had different codon responses in ribosome binding assays. The tRNA(arg) eluting first (prevalent in shaken cultures) bound strongly to polyadenylic-guanylic acid [poly(A,G)] and to a lesser extent to polycytidylic-guanylic-adenylic acid [poly(C,G,A)]. The second tRNA(arg) species (prevalent in unshaken cultures) bound to poly(C,G,A) but not to poly(A, G). The possible significance of these observations is briefly discussed. Several modifications that improve the yield of tRNA from Neurospora were introduced in a standard isolation procedure.

Amino Acids↗
PubMed1969

Synthetic spectroscopic models related to coenzymes and base pairs. IV. Stacking interactions in tRNA; the anticodon-adjacent base.

In order to test the Fuller and Hodgson hypothesis that modification of the anticodon-adjacent base in certain tRNA's not only prevents mRNA base-pairing at that site but also increases the stabilization of a stacked conformation in the anticodon loop, we have examined the interaction between adenosine and its N(6)-isopentenyl derivative by means of model compounds. The synthetic 9-[3-(aden-9-yl)propyl]-6-(3-methyl-2-butenylamino)purine, Ad-C(3)-iPAd (IV), in which the adenine and N(6)-substituted adenine moieties are joined at the 9 and 9' positions by a trimethylene chain, served as a useful spectroscopic model for assessing the base-base interaction free from the complicating features of the carbohydrate and phosphodiester groupings. The hypochromism for the model, which was determined in dilute aqueous solution and represents the decrease in integrated ultraviolet absorption intensity of Ad-C(3)-iPAd (IV) compared with equimolar 9-propyladenine (Ad-C(3)) and 6-(3-methyl-2-butenyl-amino)-9-propylpurine (V, iPAd-C(3)), was 17.9 per cent in neutral solution, 8.4 per cent in 0.1 N HCl, and 18.5 per cent in 0.1 N NaOH. Comparison with the per cent hypochromism calculated for the simple model Ad-C(3)-Ad (e.g., 16.5% in neutral solution) confirms the strong interaction in IV observed between uncharged plane-parallel adenine and N(6)-substituted adenine rings. The cause for changes in the absorption spectrum of Ad-C(3)-iPAd are discussed. The fluorescence and phosphorescence emission spectra of Ad-C(3)-iPAd in ethylene glycol-water glass at 80 degrees K add considerable weight to the conclusion that there is a strong tendency for adenine and N(6)-(Delta(2)-isopentenyl)adenosine (I) to stack if this is permitted by steric considerations.

Adenine Nucleotides↗
PubMed2001

1-Methylguanosine in place of Y base at position 37 in phenylalanine tRNA is responsible for its shiftiness in retroviral ribosomal frameshifting.

Many mammalian retroviruses express their protease and polymerase by ribosomal frameshifting. It was originally proposed that a specialized shifty tRNA promotes the frameshift event. We previously observed that phenylalanine tRNA(Phe) lacking the highly modified wybutoxosine (Y) base on the 3' side of its anticodon stimulated frameshifting, demonstrating that this tRNA is shifty. We now report the shifty tRNA(Phe) contains 1-methylguanosine (m(1)G) in place of Y and that the m(1)G form from rabbit reticulocytes stimulates frameshifting more efficiently than its m(1)G-containing counterpart from mouse neuroblastoma cells. The latter tRNA contains unmodified C and G nucleosides at positions 32 and 34, respectively, while the former tRNA contains the analogous 2'-O-methylated nucleosides at these positions. The data suggest that not only does the loss of a highly modified base from the 3' side of the anticodon render tRNA(Phe) shifty, but the modification status of the entire anticodon loop contributes to the degree of shiftiness. Possible biological consequences of these findings are discussed.

Animals↗
PubMed1994

A nuclear tRNA gene cluster in the protozoan Leishmania tarentolae and differential distribution of nuclear-encoded tRNAs between the cytosol and mitochondria.

All mitochondrial tRNAs in the protozoan Leishmania are believed to be encoded in the nuclear genome and imported selectively into the mitochondria by an as yet unknown mechanism. Previously, we reported that two tRNAs whose genes are tightly linked were imported by mitochondria. In contrast, a tRNA encoded by a lone tRNA gene was not detectable in mitochondria. The lone tRNA gene had flanking sequences that were different from the linked genes. These studies implied a possible correlation between tRNA gene organization and gene flanking sequence, and selective tRNA import into mitochondria. Here, we report the identification of a cluster of 10 tRNA genes and show the distribution of the corresponding tRNAs in cytosolic and mitochondrial fractions. tRNA(leu)(CAG) and tRNA2(arg)(TCG) are abundant in the cytosol, but relatively scarce in mitochondria. Conversely, tRNA(ile)(TAT) and tRNA1(lys)(TTT) are abundant in mitochondria, but relatively scarce in the cytosol. tRNA(val)(TAC) and tRNA2(thr)(TGT) are barely detectable in either cellular compartment, while tRNA(gln)(TTG), tRNA1(arg)(ACG), tRNA(gly)(TCC), and tRNA(trp)(CCA) are detected in approximately equal levels in both compartments. Sequencing of the 2600 bp that comprise the tRNA gene cluster also encoding the genes for 5S RNA and URNAB RNA indicates that nucleotide composition, length, and location of genes within the cluster do not clearly correlate with import characteristics. The unexpected presence of the tRNA(trp)(CCA)-gene transcript in mitochondria is also reported. Evidence suggests that this tRNA may have unidentified base modifications at the anticodon triplet.

Animals↗
PubMed2004

Crystal structure of the apo forms of psi 55 tRNA pseudouridine synthase from Mycobacterium tuberculosis: a hinge at the base of the catalytic cleft.

The three-dimensional structure of the RNA-modifying enzyme, psi55 tRNA pseudouridine synthase from Mycobacterium tuberculosis, is reported. The 1.9-A resolution crystal structure reveals the enzyme, free of substrate, in two distinct conformations. The structure depicts an interesting mode of protein flexibility involving a hinged bending in the central beta-sheet of the catalytic module. Key parts of the active site cleft are also found to be disordered in the substrate-free form of the enzyme. The hinge bending appears to act as a clamp to position the substrate. Our structural data furthers the previously proposed mechanism of tRNA recognition. The present crystal structure emphasizes the significant role that protein dynamics must play in tRNA recognition, base flipping, and modification.

Binding Sites↗
PubMed1982

Changes of post-transcriptional modification of wye base in tumor-specific tRNAPhe.

Nucleotide sequences of normal mouse liver tRNAPhe and tumor-specific tRNAPhes isolated from Ehrlich ascites tumor and neuroblastoma cells were examined by post-labeling techniques. The results showed that their sequences are identical, except for changes in post-transcriptional modifications that are located in the anticodon region. Normal mouse liver tRNAPhe contained Cm32, Gm34 and YOH37. On the other hand, tumor-specific tRNAPhes were found in one of two possible configurations: 1) Cm32, Gm34 and Y*OH37 (under-modified YOH) or 2) C32, G34 and m1G37. The ratio of the two forms of tRNAPhes differed in different tumor cells; Ehrlich ascites tumor tRNAPhe had mainly Y*OH-containing tRNAPhe whereas neuroblastoma tRNAPhe has predominantly m1G-containing tRNAPhe. It was concluded that tumor-specific tRNAPhes are products of different extents of modification, rather than of new tRNA transcription.

Animals↗
PubMed1996

Quantitative measurement of dihydrouridine in RNA using isotope dilution liquid chromatography-mass spectrometry (LC/MS).

A method has been developed for the microscale determination of 5,6-dihydrouridine, the most common post-transcriptional modification in bacterial and eukaryotic tRNA. The method is based on stable isotope dilution liquid chromatography-mass spectrometry (LC/MS) using [1,3-15N2]dihydrouridine and [1,3-15N2]uridine as internal standards. RNA samples were enzymatically digested to nucleosides before addition of the internal standards and subsequently analyzed by LC/MS with selected ion monitoring of protonated molecular ions of the labeled and unlabeled nucleosides. Sample quantities of approximately 1 pmol tRNA and 5 pmol 23S rRNA were analyzed for mole% dihydrouridine. Dihydrouridine content of Escherichia coli tRNASer(VGA) and tRNAThr(GGU) as controls were measured as 2.03 and 2.84 residues/tRNA molecule, representing accuracies of 98 and 95%. Overall precision values for the analyses of E. coli tRNASer(VGA) and E. coli tRNAThr(GGU), unfractionated tRNA from E. coli and 23S rRNA from E. coli were within the range 0.43-2.4%. The mole% dihydrouridine in unfractionated tRNA and 23S rRNA from E. coli were determined as 1.79 and 0.0396%, corresponding to 1.4 and 1.1 residues/RNA molecule respectively.

Chromatography, High Pressure Liquid↗
PubMed1981

Yeast ochre suppressor SUQ5-ol is an altered tRNA Ser UCA.

Ochre suppressor tRNA was partially purified from strains of Saccharomyces cerevisiae containing the serine-inserting class III suppressor SUQ5-ol. RNA sequence analysis of this tRNA indicated that the suppressor is derived from a UCA-decoding tRNA Ser by a G leads to U substitution in the middle position of the anticodon. The suppressor further differs from the wild-type UCA-decoding tRNA Ser in that the mutant anticodon lacks the modified uridine found in the wobble position of the wild-type tRNA and contains instead another modification in or near the anticodon.

Anticodon↗
PubMed2006

Sno/scaRNAbase: a curated database for small nucleolar RNAs and cajal body-specific RNAs.

Small nucleolar RNAs (snoRNAs) and Cajal body-specific RNAs (scaRNAs) are named for their subcellular localization within nucleoli and Cajal bodies (conserved subnuclear organelles present in the nucleoplasm), respectively. They have been found to play important roles in rRNA, tRNA, snRNAs, and even mRNA modification and processing. All snoRNAs fall in two categories, box C/D snoRNAs and box H/ACA snoRNAs, according to their distinct sequence and secondary structure features. Box C/D snoRNAs and box H/ACA snoRNAs mainly function in guiding 2'-O-ribose methylation and pseudouridilation, respectively. ScaRNAs possess both box C/D snoRNA and box H/ACA snoRNA sequence motif features, but guide snRNA modifications that are transcribed by RNA polymerase II. Here we present a Web-based sno/scaRNA database, called sno/scaRNAbase, to facilitate the sno/scaRNA research in terms of providing a more comprehensive knowledge base. Covering 1979 records derived from 85 organisms for the first time, sno/scaRNAbase is not only dedicated to filling gaps between existing organism-specific sno/scaRNA databases that are focused on different sno/scaRNA aspects, but also provides sno/scaRNA scientists with an opportunity to adopt a unified nomenclature for sno/scaRNAs. Derived from a systematic literature curation and annotation effort, the sno/scaRNAbase provides an easy-to-use gateway to important sno/scaRNA features such as sequence motifs, possible functions, homologues, secondary structures, genomics organization, sno/scaRNA gene's chromosome location, and more. Approximate searches, in addition to accurate and straightforward searches, make the database search more flexible. A BLAST search engine is implemented to enable blast of query sequences against all sno/scaRNAbase sequences. Thus our sno/scaRNAbase serves as a more uniform and friendly platform for sno/scaRNA research. The database is free available at http://gene.fudan.sh.cn/snoRNAbase.nsf.

Base Sequence↗
PubMed1983

[In vitro inhibition of DNA replication by local anesthetics. Effects on human MCF7 neoplastic cells].

The action of two local anesthetics (Lidocaine and Bupivacaine) on cells of mammary carcinoma MCF7 was investigated. 3H-TdR incorporation decreases in relation to the dose, and viability by Trypan blue does not significantly change but at high doses of anesthetic. Intercell adhesion decreases only at high concentration. When Lidocaine is removed after the fourth hour and Bupivacaine after the second hour the antimitotic action is irreversible. The inhibiting action of drugs is related to the cell number and unrelated to the time of adding the drug. There was no change of Lidocaine and Bupivacaine action on neoplastic cells at different concentration of Na+, K+ and Ca++ in the medium. Neoplastic cells are partially independent from Ca++ and we think the antimitotic effect of local anesthetics we observed can be due to: antagonist action to calmodulin; inhibition of aminoacylation of tRNA; inhibition of cholesterol synthesis; modification of membrane permeability which is however significant only for high concentration of the drug.

Breast Neoplasms↗
PubMed1981

Lack of a specific ribose methylation at guanosine 17 in Morris hepatoma 5123D tRNASer1IGA.

Tumor transfer RNA's (tRNA's) frequently exhibit alterations in column chromatographic profiles and in base compositions when compared to their normal counterparts. Because such alterations may be involved in the dedifferentiated state of cancer cells, it is of interest to determine their structural basis and functional significance. The recent development of highly sensitive postlabeling methods has now made possible sequence analysis of tRNA's from neoplastic tissues available only in limited amounts. We have determined the nucleotide sequence of Morris hepatoma serine tRNA (anticodon IGA) and compared it with its normal counterpart in rat liver. The tumor serine tRNA was found to lack the ribose methylation of guanosine in position 17 of the dihydrouridine loop present in the liver RNA. This result explains the column chromatographic shifts of Morris hepatoma 5123D seryl-tRNA isoacceptors, suggesting that all seryl-tRNA isoacceptors may lack this modification.

Animals↗
PubMed1982

Lysine tRNAs from Bacillus subtilis 168: structural analysis.

The primary sequence was established for two lysine tRNA isoacceptors which differ in abundance during development in Bacillus subtilis. Both tRNAs shared the same primary sequence but differed in the degree of post-transcriptional modification in the anticodon loop. The earlier eluting species, tRNA lys 1, had an unmodified C in position 32 and a mixture of N-[9-beta-ribofuranosyl) purin-6-ylcarbamoyl]-L-threonine, t6A, and N-[(9-beta-D-ribofuranosyl-2-methylthio-purin-6-yl)carbamoyl]threonine, ms2t6A, in position 37. The later eluting species, tRNA Lys 3, which is the more efficient in protein synthesis, had a modified C in position 32 and only ms2t6A in position 37. The possibility exists that modification to make a more efficient tRNA species may be part of a functional interaction between the translational and transcriptional changes that are part of the differentiation process in B. subtilis.

Anticodon↗
PubMed2000

A pathogenic point mutation reduces stability of mitochondrial mutant tRNA(Ile).

Point mutations in mitochondrial tRNA genes are responsible for individual subgroups of mitochondrial encephalomyopathies. We have recently reported that point mutations in the tRNA(Leu)(UUR) and tRNA(Lys) genes cause a defect in the normal modification at the first nucleotide of the anticodon. As part of a systematic analysis of pathogenic mutant mitochondrial tRNAs, we purified tRNA(Ile) with a point mutation at nucleotide 4269 to determine its nucleotide sequence, including modified nucleotides. We found that, instead of causing a defect in the post-transcriptional modification, a pathogenic point mutation in the mitochondrial tRNA(Ile) reduced the stability of the mutant tRNA molecule, resulting in a low steady-state level of aminoacyl-tRNA. The reduced stability was confirmed by examining the life-span of the mutant tRNA(Ile) both in vitro and in vivo, as well as by monitoring its melting profile. Our finding indicates that the mutant tRNA(Ile) itself is intrinsically unstable.

Acylation↗
PubMed1978

Chemical modification as a probe of conformational changes in transfer ribonucleic acid on aminoacylation.

Treatment of Escherichia coli CA265 phenylalanyl-tRNA with 3M-NaHSO3, pH6.0, at 25 degrees C resulted in modification of four bases and in the deacylation of the charged tRNAphe. The similarity of the rates of base modification and of the deacylation of the phenylalanyl-tRNA permitted the isolation of partially modified phenylalanyl-tRNAphe and partially modified deacylated tRNAphe. The sites and extents of base modification in these fractions were determined and found to be the same as those in uncharged tRNAphe modified under identical conditions. These findings are discussed in relation to previous evidence for and against a conformational change in tRNA on its aminoacylation. The methods described should prove adaptable to study of other aminoacyl-tRNA species.

Chemical Phenomena↗
PubMed2004

A pathogenesis-associated mutation in human mitochondrial tRNALeu(UUR) leads to reduced 3'-end processing and CCA addition.

Point mutations in mitochondrial tRNAs can cause severe multisystemic disorders such as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and myoclonus epilepsy with ragged-red fibers (MERRF). Some of these mutations impair one or more steps of tRNA maturation and protein biosynthesis including 5'-end-processing, post-transcriptional base modification, structural stability, aminoacylation, and formation of tRNA-ribosomal complexes. tRNALeu(UUR), an etiologic hot spot for such diseases, harbors 20 of more than 90 disease-associated mutations described to date. Here, the pathogenesis-associated base substitutions A3243G, T3250C, T3271C, A3302G and C3303T within this tRNA were tested for their effects on endonucleolytic 3'-end processing and CCA addition at the tRNA 3'-terminus. Whereas mutations A3243G, A3302G and C3303T reduced the efficiency of 3'-end cleavage, only the C3303T substitution was a less efficient substrate for CCA addition. These results support the view that pathogenesis may be elicited through cumulative effects of tRNA mutations: a mutation can impede several pre-tRNA processing steps, with each such reduction contributing to the overall impairment of tRNA function.

Humans↗
PubMed1998

Presence and location of modified nucleotides in Escherichia coli tmRNA: structural mimicry with tRNA acceptor branches.

Escherichia coli tmRNA functions uniquely as both tRNA and mRNA and possesses structural elements similar to canonical tRNAs. To test whether this mimicry extends to post-transcriptional modification, the technique of combined liquid chromatography/ electrospray ionization mass spectrometry (LC/ESIMS) and sequence data were used to determine the molecular masses of all oligonucleotides produced by RNase T1 hydrolysis with a mean error of 0.1 Da. Thus, this allowed for the detection, chemical characterization and sequence placement of modified nucleotides which produced a change in mass. Also, chemical modifications were used to locate mass-silent modifications. The native E.coli tmRNA contains two modified nucleosides, 5-methyluridine and pseudouridine. Both modifications are located within the proposed tRNA-like domain, in a seven-nucleotide loop mimicking the conserved sequence of T loops in canonical tRNAs. Although tmRNA acceptor branches (acceptor stem and T stem-loop) utilize different architectural rules than those of canonical tRNAs, their conformations in solution may be very similar. A comparative structural and functional analysis of unmodified tmRNA made by in vitro transcription and native E.coli tmRNA suggests that one or both of these post-transcriptional modifications may be required for optimal stability of the acceptor branch which is needed for efficient aminoacylation.

Base Composition↗
PubMed1975

Reaction heat variation with pH in formation of the trypsin-soybean inhibitor complex.

The heat of reaction between beta-trypsin and Kunitz soybean inhibitor (STI) hasbeen measured at 5 degrees and 25 degrees from pH 4 to 8.5. Corresponding measuremenportion of tRNA-Gly2-GGA/G molecules isolated from E. coli cells. The missense suppressor mutation, glyTsuA36(HA), results in a C yields U base substitution at the 3' end of the anticodon of tRNA-Gly2-GGA/G(nucleotide position 38). Asecondary effect of this base substitution is the modification of the A residue directly adjacent to the 3' end of the anticodon of tRNA-Gly2-suA36(HA), suggesting that the enzymes responsible for this modification recognize the anticodon sequencesof prospective tRNA substrates. The creation of a missense-suppressing tRNA, tRNA-Gly2-suA36(HA), by an alteration of the anticodon sequence of tRNA-Gly2-GGA/G is analogous to mechanisms whereby other suppressor tRNAs have arisen. The high degree of nucleotide sequence homology between the amino acid acceptor stems and anticodon regions may be recognized by the glycyl-tRNA synthetase; the involvement of theanticodon region in the synthetase recognition process is supported by the greatly decreased rate of aminoacylation of tRNA-Gly2-suA36(HA).

Hydrogen-Ion Concentration↗
PubMed2005

tRNA m7G methyltransferase Trm8p/Trm82p: evidence linking activity to a growth phenotype and implicating Trm82p in maintaining levels of active Trm8p.

We show that Saccharomyces cerevisiae strains lacking Trm8p/Trm82p tRNA m7G methyltransferase are temperature-sensitive in synthetic media containing glycerol. Bacterial TRM8 orthologs complement the growth defect of trm8-Delta, trm82-Delta, and trm8-Delta trm82-Delta double mutants, suggesting that bacteria employ a single subunit for Trm8p/Trm82p function. The growth phenotype of trm8 mutants correlates with lack of tRNA m7G methyltransferase activity in vitro and in vivo, based on analysis of 10 mutant alleles of trm8 and bacterial orthologs, and suggests that m7G modification is the cellular function important for growth. Initial examination of the roles of the yeast subunits shows that Trm8p has most of the functions required to effect m7G modification, and that a major role of Trm82p is to maintain cellular levels of Trm8p. Trm8p efficiently cross-links to pre-tRNAPhe in vitro in the presence or absence of Trm82p, in addition to its known residual tRNA m7G modification activity and its SAM-binding domain. Surprisingly, the levels of Trm8p, but not its mRNA, are severely reduced in a trm82-Delta strain. Although Trm8p can be produced in the absence of Trm82p by deliberate overproduction, the resulting protein is inactive, suggesting that a second role of Trm82p is to stabilize Trm8p in an active conformation.

Amino Acid Sequence↗