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PubMed1977

Enzymatic incorporation of ATP and CTP analogues into the 3' end of tRNA.

Structural analogues of adenosine 5'-triphosphate and cytidine 5'-triphosphate were investigated as substrates for ATP(CTP):tRNA nucleotidyl transferase. Eight out of 26 ATP analogues and six out of nine CTP analogues were incorporated into the 3' terminus of tRNA. In general, for the recognition of the substrates the modification of the cytidine is less critical than is the modification of adenosine. An isosteric substitution on the ribose residue is possible in both CTP and ATP. The free hydroxyls of these triphosphates can be replaced by an amino group or hydrogen atom without loss of substrate properties. Modifications of positions 1, 2, 6, and 8 on the adenine ring of ATP are not allowed whereas modification on positions 2, 4 and 5 on the cytosine ring of CTP are tolerated by the enzyme. No differences can be observed in the substrate properties of ATP(CTP):tRNA nucleotidyl transferase isolated from different sources. Methods for preparation of tRNA species, which are shortened at their 3' end by one or more nucleotides, and analytical procedures for characterisation of these modified tRNAs are described.

Adenosine Triphosphate↗
PubMed1975

The effect of modification of T7 DNA by the carcinogen N-1-acetylaminofluorene: termination of transcription in vitro.

To study the effects of N-2-acetylaminofluorene (AAF) modification of DNA on transcription, purified DNA from bacteriophage T7 was modified in vitro to varying extent with AAF and transcribed by DNA-dependent RNA polymerase from Escherichia coli. The main effects of AAF modification on transcription are a marked inhibition of the rate and extent of trna synthesis with relatively little effect on initiation except at very high AAF doses. Calibration of the percent modification with [14-C]AAF and analysis of the size of the RNA product by double isotope labeling and polyacrylamide gel electrophoresis support the following mechanism of transcription inhibition: most of the AAF residues bound to the coding strand of the DNA cause premature termination of transcription, at or near the site of modification, with release of RNA polymerase. This results in the production of shorter RNA chains with increasing amounts of bound carcinogen. The data are consistent with there being no reinitiation and/or synthesis of RNA distal to the AAF-modification site.

Carcinogens↗
PubMed1988

Interaction of tRNAs and of phosphorothioate-substituted nucleic acids with an organomercurial. Probing the chemical environment of thiolated residues by affinity electrophoresis.

The interactions of 4-thiouridine and 5-[(methylamino)methyl]-2-thiouridine in tRNA and of phosphorothioate esters in nucleic acids with an organomercurial have been investigated. For this purpose, an affinity electrophoretic system has been developed in which the mercury derivative has been covalently immobilized in a standard polyacrylamide gel. The retardation of thiolated macromolecules was found to be sensitive to the chemical environment of the sulfur atom, giving characteristic interaction constants dependent on the nature of the modification and its accessibility to binding. The interaction could, in the case of tRNA, be abolished by conventional specific chemical modification of the thiolated bases, as well as by irradiation with 32P-derived beta-emission. Not only has the fractionation of sulfur-modified from unmodified species been attained but a quantitative application of the technique has made it possible to study the binding of mercury and, by competition, that of magnesium in terms of the conformation of tRNA.

Autoradiography↗
PubMed1980

The nucleus as the site of tRNA methylation.

Mouse L-cells were enucleated by exposure to cytochalasin B followed by centrifugation. The resulting karyoplasts, nuclei surrounded by a thin shell of cytoplasm and an outer cell membrane, and cytoplasts, the enucleated cell cytoplasm, were assayed for tRNA methyltransferase activity. The bulk of the enzyme activity was found to be localized in the nuclei. Analysis of the methylated nucleosides produced by the enzyme from the two sources showed that all the base-specific enzyme activities which are found in whole cell extracts were present in the nuclear extracts. The cytoplast extracts retained a low but detectable enzyme activity, which was composed predominantly of only two base-specific activities. This may represent tRNA methyltransferases of the mitochondria or may be cytoplasmic enzymes for late modification reactions.

Cell Nucleus↗
PubMed1984

Interactions between avian myeloblastosis reverse transcriptase and tRNATrp. Mapping of complexed tRNA with chemicals and nucleases.

The interactions between beef tRNATrp with avian myeloblastosis reverse transcriptase have been studied by statistical chemical modifications of phosphate (ethylnitrosourea) and cytidine (dimethyl sulfate) residues, as well as by digestion of complexed tRNA by Cobra venom nuclease and Neurospora crassa endonuclease. Results with nucleases and chemicals show that reverse transcriptase interacts preferentially with the D arm, the anticodon stem and the T psi stem. All these regions are located in the outside of the L-shaped structure of tRNA. This domain of interaction is different to that reported previously in the complex of beef tRNA with the cognate aminoacyl-tRNA synthetase (M. Garret et al.; Eur. J. Biochem. In press). Avian reverse transcriptase destabilizes the region of tRNA where most of the tertiary interactions maintaining the structure of tRNA are located.

Alkylation↗
PubMed2005

Unusual usage of wobble modifications in mitochondrial tRNAs of the nematode Ascaris suum.

To understand the decoding property of nematode mitochondrial tRNAs with unusual secondary structures, post-transcriptional modifications at wobble positions of Ascaris suum mitochondrial tRNAs corresponding to two-codon families ending with a purine were analyzed. 5-Carboxymethylaminomethyluridine (cmnm(5)U) was identified at the wobble positions of tRNA(Lys), tRNA(Glu) and tRNA(Gln), while 5-carboxymethylaminomethyl-2-thiouridine (cmnm(5)s(2)U) was present in tRNA(UAA)(Leu)andtRNA(Trp). In most bacterial and mitochondrial tRNAs, the 2-thiouridine derivative is present in tRNAs for Lys, Glu and Gln. These is no report that cmnm(5)s(2)U is used in tRNA(UAA)(Leu)andtRNA(Trp). The unusual usage of wobble modifications might assist decoding of nematode mitochondrial mRNAs.

Animals↗
PubMed2000

NMR studies of Bacillus subtilis tRNA(Trp) hyperexpressed in Escherichia coli. Assignment of imino proton signals and determination of thermal stability.

15N-Labeled Bacillus subtilis tRNA(Trp) wild type and a series of mutants were hyperexpressed in Escherichia coli and purified for NMR studies with the use of two-dimensional nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear single quantum correlation (HSQC) and three-dimensional NOESY-HSQC techniques. These made possible chemical shift assignments of imino protons and determination of the thermal stability of the tRNA(Trp) molecules. Almost all of the imino protons in the helical regions and the tertiary base pairs were assigned, except three imino protons of the AU base pairs whose peaks were not clearly observed. Several base triplets found in the crystal structure of tRNA were observed in the present study as well. These studies also revealed two components of tRNA(Trp), which could not be separated by high pressure liquid chromatography, corresponding to s(4)U and U at position 8 of the tRNA(Trp), as indicated by two different sets of peaks for the TpsiC and D arms. The modification at position 8 altered the local conformation of the core region of the tRNA. Thermal unfolding experiments showed that the unfolding process is cooperative in the presence of a high concentration of magnesium ions and that the component corresponding to the s(4)U8 is more stable than the U8 component, thus providing evidence that the thiolation of U8 stabilizes the tertiary structure of tRNA.

Anticodon↗
PubMed1986

Actions of the anticodon arm in translation on the phenotypes of RNA mutants.

In previous publications, we have shown that it is practical to study the translational activity of tRNAs by replacement and alteration of the anticodon arm sequence of the genus on a plasmid clone. Experiments in which the anticodon arm sequence is transplanted between tRNA genes suggest that the translational activity is determined by these sequences. We have therefore made every variant of the anticodon loop and the three base-pairs of the stem proximal to the loop, in order to resolve the relation between the structure of Su7Am tRNATrp, and its function. All derivatives conserved the normal secondary structure of the molecule, which was known to be essential for translational activity. The probability of translation of the amber codon by these suppressors is measured in this work. This translational activity in vivo is rationalized in terms of data on the copy numbers of the plasmid clones, the nucleotide modifications of the tRNAs, the steady-state level of the mature tRNA, and the aminoacylation of these molecules. Nucleotide modification levels vary among these tRNAs, giving information about the specificities of modification systems that make O-methylribose, pseudouridine, and modified A in the anticodon arm. However, for this series of tRNAs, none of these modifications has a strong effect on translational efficiency of the tRNAs. A few of the substitutions reduce aminoacylation of the tRNAs with glutamine, as determined by comparison of suppression in normal strains and related strains, which have 25-fold elevated levels of the glutaminyl-tRNA synthetase (GlnRS). The substitutions that have the largest effect on GlnRS action are, unexpectedly, purines for conserved pyrimidines on the 5' side of the anticodon loop. Data on the concentrations of tRNA in vivo suggest that the anticodon loop and helix contribute similarly to the determination of the steady-state level of the tRNAs. This level varies sevenfold, though all tRNAs are processed from a homologous precursor made from the same transcription unit. Effects on levels appear to be mediated by changes in anticodon arm structure. A robust equation that relates aminoacyl-tRNA levels to suppressor efficiency is developed in order to resolve effects on tRNA levels and on ribosomal steps: E = A/(K + A), where E is efficiency, A is aminoacyl-tRNA concentration, and K is the effective concentration, or cellular tRNA content required for an individual tRNA to have an efficiency of 0.50. The tRNAs vary in their intrinsic ability to function on the ribosome (represented by K), after other influences have been normalized.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acyl-tRNA Synthetases↗
PubMed1996

A tyrosyl-tRNA synthetase recognizes a conserved tRNA-like structural motif in the group I intron catalytic core.

The Neurospora crassa mitochondrial (mt) tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns, in addition to aminoacylating tRNA(Tyr). Here, we compared the CYT-18 binding sites in the N. crassa mt LSU and ND1 introns with that in N. crassa mt tRNA(Tyr) by constructing three-dimensional models based on chemical modification and RNA footprinting data. Remarkably, superimposition of the CYT-18 binding sites in the model structures revealed an extended three-dimensional overlap between the tRNA and the group I intron catalytic core. Our results provide insight into how an RNA-splicing factor can evolve from a cellular RNA-binding protein. Further, the structural similarities between group I introns and tRNAs are consistent with an evolutionary relationship and suggest a general mechanism for the evolution of complex catalytic RNAs.

Base Sequence↗
PubMed1992

Disruption of the gene for Met-tRNA(fMet) formyltransferase severely impairs growth of Escherichia coli.

In bacteria, as well as in chloroplasts and mitochondria, the free amino group of the methionylated initiator tRNA(fMet) is specifically modified by the addition of a formyl group. The importance of this modification remains unclear. With the availability of pure Escherichia coli 10-formyltetrahydrofolate:L-methionyl-tRNA(fMet) N-formyltransferase, the enzyme catalyzing Met-tRNA(fMet) formylation, the corresponding fmt gene and its flanking regions were cloned and sequenced. The chromosomal fmt gene was disrupted, and strains modified in their formylation activity were constructed. A depletion of the cellular formylation activity was accompanied by a decrease in the growth rate of the bacteria. At 37 degrees C, in a rich medium, the absence of a functional fmt gene reduced the growth rate to 0.28 doubling per h, from 2.3 for the control strain. At 42 degrees C, the studied fmt mutant strain did not grow further.

Acyltransferases↗
PubMed1999

Search for differences in post-transcriptional modification patterns of mitochondrial DNA-encoded wild-type and mutant human tRNALys and tRNALeu(UUR).

Post-transcriptional modifications are characteristic features of tRNAs and have been shown in a number of cases to influence both their structural and functional properties, including structure stabilization, amino-acylation and codon recognition. We have developed an approach which allows the investigation of the post-transcriptional modification patterns of human mitochondrial wild-type and mutant tRNAs at both the qualitative and the quantitative levels. Specific tRNA species are long-term labeled in vivo with [32P]orthophosphate, isolated in a highly selective way, enzymatically digested to mononucleotides and then subjected to two-dimensional thin layer chromatographic analysis. The wild-type tRNALysand the corresponding tRNALyscarrying the A8344G mutation associated with the MERRF (Myoclonic Epilepsy with Ragged Red Fibers) syndrome exhibit the same modified nucleotides at the same molar concentrations. By contrast, a quantitatively different modification pattern was observed between the wild-type tRNALeu(UUR)and its counterpart carrying the A3243G mutation associated with the MELAS (Mitochondrial Myopathy, Encephalopathy with Lactic Acidosis and Stroke-like episodes) syndrome, the latter exhibiting a 50% decrease in m2G content. Complementary sequencing of tRNALeu(UUR)has allowed the localization of this modification at position 10 within the D-stem of the tRNA. The decreased level of this modification may have important implications for understanding the molecular mechanism underlying the MELAS-associated mitochondrial dysfunction.

Autoradiography↗
PubMed1977

Isolation and characterization of valine transfer RNA from Saccharomyces cerevisiae.

Two procedures for isolating valine tRNA from commercial bakers' yeast were investigated. The first involved: (a) counter double current distribution; (b) chromatography on benzoyl-DEAE-cellulose; (c) reverse phase chromatography on Chromosorb G saturated with trioctylpropylammonium bromide (Oakridge System 3). The material isolated lacked the 3'-terminal adenylic acid residue. The second procedure involved the first two steps above followed by: (a) enzymatic aminoacylation with a partially purified yeast extract; (b) derivatization with N-phenoxyacetoxysuccinimide; (c) chromatography on benzoyl-DEAE-cellulose; (d) reverse phase chromatography, System 3. The product was intact tRNA. It was a mixture of isoacceptors (59:41) differing by a modification (uracil leads to dihydrouracil) at position 48. It was free of denatured material; specific activity 1,825 pmol of valine/A260 unit of tRNA. Sequence analysis confirmed the recently corrected structure (Bonnet, J., Ebel, J. P., Dirheimer, G., Shershneva, L. P., Krutilina, A. I., Venkstern, T. V., and Bayev, A. A. (1974) Biochimie 56, 1211-1213). A preliminary study of the alkaline hydrolysis of the 7-methylguanosine residue that occurs at position 47 showed that at least two products are formed instead of only one as usually quoted in the literature. A rapid, ultramicro, chromatographic system for separating these products and measuring them quantitatively is described.

Guanosine↗
PubMed1986

Effect of a 2-methylthio-N6-isopentenyladenosine deficiency on peptidyl-tRNA release in Escherichia coli.

We examined the effect of miaA, a mutation conferring a deficiency in 2-methylthio-N6-isopentenyladenosine in tRNA, on patterns of peptidyl-tRNA accumulation in Escherichia coli strains deficient in peptidyl-tRNA hydrolase activity. A specific reduction in peptidyl-tRNA accumulation was seen for tRNAs which normally contain the 2-methylthio-N6-isopentenyladenosine modification. These results provide new evidence in support of the ribosome editor model, which links peptidyl-tRNA release to mistranslation events.

Adenosine↗
PubMed1982

Chemical probes for tRNA tertiary structure. Comparative alkylation of tRNA with methylnitrosourea, ethylnitrosourea and dimethylsulfate.

The tertiary structure of tRNA in solution can be proved by chemical modification experiments. Three reagents, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea and dimethylsulfate which are known to alkylate nucleic acids at nucleophilic centers were compared. It is found that N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea mainly react with phosphate residues and dimethylsulfate only with the bases. With dimethylsulfate the extent of alkylation of guanosines is about one order of magnitude higher than that of the phosphates by the nitroso compounds.

Alkylating Agents↗
PubMed2000

The SsrA-SmpB system for protein tagging, directed degradation and ribosome rescue.

Bacteria contain a remarkable RNA molecule - known alternatively as SsrA RNA, tmRNA, or 10Sa RNA - that acts both as a tRNA and as an mRNA to direct the modification of proteins whose biosynthesis has stalled or has been interrupted. These incomplete proteins are marked for degradation by cotranslational addition of peptide tags to their C-termini in a reaction that is mediated by ribosome-bound SsrA RNA and an associated protein factor, SmpB. This system plays a key role in intracellular protein quality control and also provides a mechanism to clear jammed or obstructed ribosomes. Here the structural, functional and phylogenetic properties of this unique RNA and its associated factors are reviewed, and the intracellular proteases that act to degrade the proteins tagged by this system are also discussed.

Amino Acid Sequence↗
PubMed1997

Synthesis and studies on the effect of 2-thiouridine and 4-thiouridine on sugar conformation and RNA duplex stability.

In order to understand the effect of 2-thiouridine (s2U) substitution on RNA structure and the potential for stabilization of tRNA codon-anticodon interactions through s2U-34 modification, a pentamer RNA sequence, Gs2UUUC, was synthesized and characterized by NMR spectroscopy. The single strand contains the UUU anticodon sequence of tRNALys with flanking GCs to increase duplex stability. Regiochemical effects of uridine thiolation were determined by comparing the structure and stability of the 2-thiouridine containing oligonucleotide with an identical sequence containing 4-thiouridine (s4U) and also the normal uridine nucleoside. Circular dichroism spectrum indicated an A-form helical conformation for Gs2UUUC which was further confirmed by 2D ROESY NMR experiments. The duplex stability of the three pentamers complexed with a 2'-O-methyl-ribonucleotide complementary strand, GmAmAmAmCm, was determined by UV thermal melting studies and by 1H NMR spectroscopy. The duplex containing s2U has a T m of 30.7 degrees C compared to 19. 0 degrees C for the unmodified control and 14.5 degrees C for the s4U containing duplex. The results from UV experiments were corroborated by imino proton NMR studies that show proton exchange rates, chemical shift differences, and NH proton linewidths indicative of the stability order s2U >U >s4U. The magnitude of the effect of s2U in our model system is comparable to the 20 degrees C stabilization observed by Grosjean and co-workers for 2-thiolation in a codon-anticodon model system composed of two tRNAs with complementary anticodon sequences [Houssier, C., Degee, P., Nicoghosian, K. and Grosjean, H. (1988) J. Biomol. Struct. Dyn., 5, 1259-1266].

Anticodon↗
PubMed1979

Synthesis and activation of asparagine in asparagine auxotrophs of Saccharomyces cerevisiae.

L-Asparagine synthesis in Saccharomyces cerevisiae is performed by a glutamine-dependent asparagine synthetase of the type found in higher organisms. Auxotrophy for asparagine has been obtained in two classes of mutants. In class I, asparagine synthetase activity is cancelled. These mutants combine two mutations, asnA- and asnB-. Neither asnA- nor asnB- mutation alone leads to total auxotrophy. Partial auxotrophy as well as a strong decrease in enzyme activity result from asnA- mutation. No change is detectable in cells with the asnB- mutationalone. This, and Jones' report [J. Bacteriol. 134, 200-207 (1978)] of auxotrophy resulting from the combination of two mutations, are strong supports for asparagine synthesis being an unusual biosynthetic operation. In class II, auxotrophy results from a single mutation which leads to a modification of the efficiency of the asparaginyl-tRNA synthetase (asnRS- mutation). This auxotrophy is cancelled if asparaginase I activity (the only one present in sigma 1278b wild type) is cancelled by casnI- mutation. This latter mutation allows an increase in the asparagine pool which is able to compensate for the asparaginyl-tRNA synthetase partial defect of the asnRS- mutant.

Amino Acyl-tRNA Synthetases↗