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PubMedDate not supplied

[Photoaffinity modification of Escherichia coli ribosomes by fMet-tRNAf Met derivatives in the 70S initiation complex].

Photoaffinity labeling of E. coli ribosomes within the 70S initiation complex was studied by using photoreactive derivatives of fMet-tRNAfMet bearing arylazidogroups scattered statistically over guanosine residues. It is shown that fMet-azido-tRNAfMet-II bearing 2 moles of the reagent residues per mole of tRNA (modified in the conditions of stability of tRNA tertiary structure) is fully active in aminoacylation and in the factor-dependent binding with ribosomes to form the 70S initiation complex. Functional activity of fMet-azido-tRNAfMet-I bearing also 2 moles of the reagent residues per mole of tRNA (but modified in conditions of lability of tRNA tertiary structure) decreases up to approximately 45% in aminoacylation and up to 70% in IF-2 X GTP-dependent binding to the ribosomes. Irradiation of complexes 70S ribosome-MS2-RNA-fMet-azido-tRNAfMet results in covalent linking of the tRNA derivative to the ribosomes. Both subunits are labeled, the 30S to a larger extent than 50S. It is shown that fMet-azido-tRNAfMet-II labels proteins S1, S7, S9, L27 whereas fMet-azido-tRNAfMet-1--proteins S1, S3, S5, S9, S14, L1, L2, L7/L12.

Affinity Labels↗
PubMed1994

On the conformational stability of oligonucleotide duplexes and tRNA molecules.

Thermodynamic experiments provide a wealth of data about the conformational stability, viz., the free energy difference (delta G) between folded and unfolded states of DNA/RNA duplexes. However, there is no acceptable view about how the various non-covalent forces contribute individually to the observed stability. In particular, the role of the hydrophobic force is not clearly known. In this paper we quantitatively enumerate the stability factors, hydrogen bonding, base stacking, van der Waals, electrostatic, and hydrophobic interactions from the knowledge of the crystal structures of 15 DNA/RNA duplexes and two tRNA molecules, and translate them into free energy contributions to the stability of nucleic acid systems. Taking the experimental delta G values and computed component free energy terms for a set of duplexes, we set up multiple regression equations to predict their stabilities. After back-check and validity tests, we apply this model to predict delta G values for a large number of duplexes and two tRNA molecules (tRNAphe and tRNAasp). There is excellent agreement between the theoretical predictions and experimental observations. The considered duplexes with four to 16 base-pairs and the tRNA molecules have delta G values in a narrow range, 5-20 kcal mol-1, a range seen in a variety of globular proteins. There is no relationship between delta G and N, the number of nucleotides in the molecule. Base-stacking, hydrogen bonding and van der Waals factors contribute significantly, whereas hydrophobic and electrostatic factors contribute, respectively, marginally and minimally. The major factor which gives sequence specificity is base-stacking. The new set of atomic solvation parameters (ASPs) derived to estimate hydrophobic free energy brings to light the dangers of using already available ASPs, which emphasize the role of the hydrophobic factor unrealistically.

Base Sequence↗
PubMed1996

The turnip yellow mosaic virus tRNA-like structure cannot be replaced by generic tRNA-like elements or by heterologous 3' untranslated regions known to enhance mRNA expression and stability.

The tRNA-like structure (TLS) at the 3' end of the turnip yellow mosaic virus genome was replaced with heterologous tRNA-like elements, and with a poly(A) tail, in order to assess its role. Replacement with the valylatable TLSs from two closely related tymoviruses resulted in infectious viruses. In contrast, no systemic symptoms on plants, and only low viral accumulations in protoplasts, were observed for three chimeric genomes with 3' sequences known to enhance mRNA stability and translatability. One of these chimeras had a poly(A) tail, and the others had the TLS with associated upstream pseudoknot tracts from the 3' ends of brome mosaic and tobacco mosaic viruses. The latter two chimeric RNAs were shown to be appropriately folded by demonstrating their aminoacylation in vitro with tyrosine and histidine, respectively. The results show that enhancement of genome stability or gene expression is not the major role of the turnip yellow mosaic virus TLS. The major role is likely to be replicational, dependent on features present in tymoviral TLSs but not in generic tRNA-like structures.

Base Sequence↗
PubMed1992

A mutation in the tRNA nucleotidyltransferase gene promotes stabilization of mRNAs in Saccharomyces cerevisiae.

To identify trans-acting factors involved in mRNA decay in the yeast Saccharomyces cerevisiae, we have begun to characterize conditional lethal mutants that affect mRNA steady-state levels. A screen of a collection of temperature-sensitive mutants identified ts352, a mutant that accumulated moderately stable and unstable mRNAs after a shift from 23 to 37 degrees C (M. Aebi, G. Kirchner, J.-Y. Chen, U. Vijayraghavan, A. Jacobson, N.C. Martin, and J. Abelson, J. Biol. Chem. 265:16216-16220, 1990). ts352 has a defect in the CCA1 gene, which codes for tRNA nucleotidyltransferase, the enzyme that adds 3' CCA termini to tRNAs (Aebi et al., J. Biol. Chem., 1990). In a shift to the nonpermissive temperature, ts352 (cca1-1) cells rapidly cease protein synthesis, reduce the rates of degradation of the CDC4, TCM1, and PAB1 mRNAs three- to fivefold, and increase the relative number of ribosomes associated with mRNAs and the overall size of polysomes. These results were analogous to those observed for cycloheximide-treated cells and are generally consistent with models that invoke a role for translational elongation in the process of mRNA turnover.

Cycloheximide↗
PubMed1999

Tryptamine-mediated stabilization of tryptophanyl-tRNA synthetase in human cervical carcinoma cell line.

Tryptamine is an endogenous neuroactive metabolite of tryptophan. Interpretation of the function of this bioamine, however, is restricted to manipulation with tryptamine synthetic pathways. Meanwhile, tryptamine is a potent inhibitor of protein biosynthesis, via the competitive inhibition of tryptophanyl-tRNA synthetase (TrpRS). The influence of the persistent tryptamine inhibition on the half-life and cellular content of TrpRS was examined by chase labeling of HeLa cells and the tryptamine-resistant subline with [35S]methionine. The results indicate that long-term tryptamine treatment of HeLa cells led to a significant increase in the half-life of TrpRS while the content, in vivo phosphorylation and gene dose of TrpRS were unchanged. These findings suggest that survival of drug-resistant cells may not be due to TrpRS gene amplification, but to stabilization of TrpRS. It was shown that tryptamine is an effective inhibitor of HeLa cell growth. In contrast to the well-characterized antineoplastic compounds, conferring a many hundred-fold elevated drug resistance to tumor cells, resistance to tryptamine at very low levels was difficult to achieve, i.e. the 2-fold resistant subline was selected after 19 months of treatment of HeLa cells with gradually increasing concentrations of tryptamine. The tryptamine-resistant HeLa subline exhibited a slower growth rate than the original HeLa line when similar concentrations of both cell populations were seeded on the plates. A low tryptamine resistance and a lack of TrpRS gene amplification were observed in two tryptamine-resistant HeLa sublines and three Chinese hamster sublines. The role of TrpRS in oncogenesis and the perspective for tryptamine as a potential anti-cancer drug are discussed.

Animals↗
PubMed1999

Transfer RNA identity contributes to transition state stabilization during aminoacyl-tRNA synthesis.

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs ensure both accurate RNA recognition and the efficient catalysis of aminoacylation. The effects of tRNA(Trp)variants on the aminoacylation reaction catalyzed by wild-type Escherichia coli tryptophanyl-tRNA synthe-tase (TrpRS) have now been investigated by stopped-flow fluorimetry, which allowed a pre-steady-state analysis to be undertaken. This showed that tRNA(Trp)identity has some effect on the ability of tRNA to bind the reaction intermediate TrpRS-tryptophanyl-adenylate, but predominantly affects the rate at which trypto-phan is transferred from TrpRS-tryptophanyl adenylate to tRNA. Use of the binding ( K (tRNA)) and rate constants ( k (4)) to determine the energetic levels of the various species in the aminoacylation reaction showed a difference of approximately 2 kcal mol(-1)in the barrier to transition state formation compared to wild-type for both tRNA(Trp)A-->C73 and. These results directly show that tRNA identity contributes to the degree of complementarity to the transition state for tRNA charging in the active site of an aminoacyl-tRNA synthetase:aminoacyl-adenylate:tRNA complex.

Adenosine Monophosphate↗
PubMed2001

Post-transcriptional modification in archaeal tRNAs: identities and phylogenetic relations of nucleotides from mesophilic and hyperthermophilic Methanococcales.

Post-transcriptional modifications in archaeal RNA are known to be phylogenetically distinct but relatively little is known of tRNA from the Methanococci, a lineage of methanogenic marine euryarchaea that grow over an unusually broad temperature range. Transfer RNAs from Methanococcus vannielii, Methanococcus maripaludis, the thermophile Methanococcus thermolithotrophicus, and hyperthermophiles Methanococcus jannaschii and Methanococcus igneus were studied to determine whether modification patterns reflect the close phylogenetic relationships inferred from small ribosomal subunit RNA sequences, and to examine modification differences associated with temperature of growth. Twenty-four modified nucleosides were characterized, including the complex tricyclic nucleoside wyosine characteristic of position 37 in tRNA(Phe) and known previously only in eukarya, plus two new wye family members of presently unknown structure. The hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, reported previously only in bacterial tRNA at the first position of the anticodon, was identified by liquid chromatography-electrospray ionization mass spectrometry in four of the five organisms. The ribose-methylated nucleosides, 2'-O-methyladenosine, N(2),2'-O-dimethylguanosine and N(2),N(2),2'-O-trimethylguanosine, were found only in hyperthermophile tRNA, consistent with their proposed roles in thermal stabilization of tRNA.

Chromatography, High Pressure Liquid↗
PubMed2001

Phosphorylation of the Saccharomyces cerevisiae La protein does not appear to be required for its functions in tRNA maturation and nascent RNA stabilization.

An abundant nuclear phosphoprotein, the La autoantigen, is the first protein to bind all newly synthesized RNA polymerase III transcripts. Binding by the La protein to the 3' ends of these RNAs stabilizes the nascent transcripts from exonucleolytic degradation. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the La protein is required for the normal pathway of tRNA maturation. Experiments in which the human protein was expressed in S. pombe have suggested that phosphorylation of the La protein regulates tRNA maturation. To dissect the role of phosphorylation in La protein function, we used mass spectrometry to identify three sites of serine phosphorylation in the S. cerevisiae La protein Lhp1p. Mutant versions of Lhp1p, in which each of the serines was mutated to alanine, were expressed in yeast cells lacking Lhp1p. Using two-dimensional gel electrophoresis, we determined that we had identified and mutated all major sites of phosphorylation in Lhp1p. Lhp1p lacking all three phosphorylation sites was functional in several yeast strains that require Lhp1p for growth. Northern blotting revealed no effects of Lhp1p phosphorylation status on either pre-tRNA maturation or stabilization of nascent RNAs. Both wild-type and mutant Lhp1 proteins localized to both nucleoplasm and nucleoli, demonstrating that phosphorylation does not affect subcellular location. Thus, although La proteins from yeast to humans are phosphoproteins, phosphorylation does not appear to be required for any of the identified functions of the S. cerevisiae protein.

Amino Acid Sequence↗
PubMed2004

Conformational changes of the small ribosomal subunit during elongation factor G-dependent tRNA-mRNA translocation.

Translocation, a coordinated movement of two tRNAs together with mRNA on the ribosome, is catalyzed by elongation factor G (EF-G). The reaction is accompanied by conformational rearrangements of the ribosome that are, as yet, not well characterized. Here, we analyze those rearrangements by restricting the conformational flexibility of the ribosome by antibiotics binding to specific sites of the ribosome. Paromomycin (Par), viomycin (Vio), spectinomycin (Spc), and hygromycin B (HygB) inhibited the tRNA-mRNA movement, while the other partial reactions of translocation, including the unlocking rearrangement of the ribosome that precedes tRNA-mRNA movement, were not affected. The functional cycle of EF-G, i.e. binding of EF-G.GTP to the ribosome, GTP hydrolysis, Pi release, and dissociation of EF-G.GDP from the ribosome, was not affected either, indicating that EF-G turnover is not coupled directly to tRNA-mRNA movement. The inhibition of translocation by Par and Vio is attributed to the stabilization of tRNA binding in the A site, whereas Spc and HygB had a direct inhibitory effect on tRNA-mRNA movement. Streptomycin (Str) had essentially no effect on translocation, although it caused a large increase in tRNA affinity to the A site. These results suggest that conformational changes in the vicinity of the decoding region at the binding sites of Spc and HygB are important for tRNA-mRNA movement, whereas Str seems to stabilize a conformation of the ribosome that is prone to rapid translocation, thereby compensating the effect on tRNA affinity.

Anti-Bacterial Agents↗
PubMed1976

[Conformational heterogeneity of tRNA, detected in the reaction of guanine bases with ketoxal].

The study of kinetic characteristics of the reaction of tRNA guanine bases with kethoxal has shown that temperature, ionic strength and Mg2+ ions, i.e. factors directly affecting the spatial structure of tRNA, influence also on its internal modification. The modification degree under stabilized spatial tRNA structure depends also on the concentration of kethoxal and is expressed in fractional values of the number of modified guanosine residues per tRNA molecule, which indicates the heterogeneity of tRNA for the modification degree. Chromatography of tRNA1 Val preparation on BD cellulose after the exhaustive modification with kethoxal under conditions of stabilized spatial structure has revealed a fraction of molecules completely resistant to the modificator, and a fraction containing differently modified tRNA molecules. tRNA heterogeneity after the reaction with kethoxal (the presence of resistant and reactive forms) indicates conformational heterogeneity of tRNA, expressed in the simultaneous presence of at least two conformer families.

Aldehydes↗
PubMed2002

Novel method for selection of tRNA-driven ribozymes with enhanced stability in mammalian cells.

Intracellular stability is a critical determinant of the activity of a ribozyme in vivo. In previous studies, we succeeded in constructing an effective system for the expression of ribozymes using the promoter of a human gene for tRNA(Val). The resultant tRNA(Val)-driven ribozymes (tRNA-ribozymes) had a half-life of approximately 100 minutes. In the present study, we established a novel system for the selection of tRNA-ribozymes that were more stable than a previously generated optimally designed tRNA-ribozyme, and we confirmed that the newly selected tRNA-ribozymes worked well. Selective pressure was applied by treating cells that expressed tRNA-ribozymes with actinomycin D, and the system yielded tRNA-ribozymes with enhanced stability. The sequences isolated after selection exhibited some similarities. Furthermore, some selected tRNA-ribozymes had almost the same activity as or higher activity than that of the optimally designed tRNA-ribozyme despite the fact that the selective pressure was not aimed at enhancing the cleavage activity. Our approach might be very useful for selection not only of ribozymes with enhanced stability but also of other functional nucleic acids in vivo.

Base Sequence↗
PubMed1982

Regulation of tRNA methyltransferase activities by spermidine and putrescine. Inhibition of polyamine synthesis and tRNA methylation by alpha-methylornithine or 1,3-diaminopropan-2-ol in Dictyostelium.

Inhibitors of polyamine synthesis (alpha-methylornithine and 1,3-diaminopropan-2-ol) were used to study the relationship between polyamine synthesis and specific methylations of tRNA in Dictyostelium discoideum during vegetative growth. Polyamine concentrations were found to be 10 mM for putrescine, 1.6 mM for spermidine and 7 mM for 1,3-diaminopropane throughout the growth stage. On treatment of growing amoebae with alpha-methylornithine or with 1,3-diaminopropan-2-ol (each at 5 mM), the syntheses of putrescine, spermidine and 1,3-diaminopropane were arrested within 4h. After polyamine synthesis had ceased, the incorporation of methyl groups into tRNA was considerably decreased under conditions that had no effect on the incorporation of uridine into tRNA, or on net syntheses of protein and of DNA. The following nucleosides in tRNA were concerned: 1 methyladenosine, 5-methylcytidine, 7-methylguanosine, 2-methylguanosine, N2N2-dimethylguanosine and 5-methyluridine (ribosylthymine). The corresponding tRNA methyltransferases, determined in Mg2+-free enzyme extracts, proved to be inactive unless polyamines were added. Putrescine and/or spermidine at concentrations of 10 mM or 1-2 mM respectively stimulate the transmethylation reaction in vitro to a maximal rate and to an optimal extent at exactly the same concentrations as found in vegetative cells. In contrast, 1,3-diaminopropane, which is formed from spermidine, does not affect the methylation of tRNA in vitro at physiological concentrations. Putrescine and/or spermidine stabilize the tRNA methyltransferases in crude extracts in the presence but not in the absence of the substrate tRNA. The results support the view that S-adenosylmethionine-dependent transmethylation reactions can be regulated by alterations of polyamine concentrations in vivo.

Diamines↗
PubMed1992

Two tRNA-binding sites in addition to A and P sites on eukaryotic ribosomes.

The interaction of tRNA with 80 S ribosomes from rabbit liver was studied using biochemical as well as fluorescence techniques. Besides the canonical A and P sites, two additional sites were found which specifically bind deacylated tRNA. One of the sites is analogous to the E site of prokaryotic ribosomes, in that binding of tRNA is labile, does not depend on codon-anticodon interaction, does not protect the anticodon loop from solvent access, and requires the presence of the 3'-terminal adenosine of the tRNA. In contrast, the stability of the tRNA complex with the second site (S site) is high. tRNA binding to the S site is also codon-independent; nevertheless, the anticodon loop is shielded from solvent access. Removal of the 3'-terminal adenosine decreases the affinity of tRNA(Phe) for the S site approximately 50-fold. tRNA(Phe) is retained at the S site during translocation and through poly(Phe) synthesis. Thus, the S site does not seem to be an intermediate site for the tRNA during the elongation cycle. Rather, the tRNA bound to the S site may allosterically modulate the function of the ribosome.

Animals↗
PubMed1996

A new method for identifying the amino acid attached to a particular RNA in the cell.

To investigate the function of tRNAs or any other aminoacylable RNAs in vivo, it is important to be able to estimate the amounts and species of aminoacylated RNAs in living cells. We have developed a method of analyzing amino acids attached to particular tRNAs obtained from cells. After the ester bond between the amino acid and the 3'-adenosine moiety of a specific aminoacyl-tRNA is stabilized by acetylation of the amino acid with [14C]acetic anhydride, the aminoacyl-tRNA can be fished out with a solid-phase-attached DNA probe. The 14C-labeled acetylamino acid is then released from the thus purified acetyl-aminoacyl-tRNAs by alkaline treatment and detected by TLC analysis.

Acetic Anhydrides↗
PubMed1985

Biochemical research on oogenesis. Aminoacyl tRNA turns over in the 42-S particles of Xenopus laevis oocytes, but its ester bond is protected against hydrolysis.

The ester bond aminoacyl tRNA is protected against hydrolysis in the 42-S particles (thesaurisomes) present in Xenopus laevis previtellogenic oocytes. Deacylation of tRNA is very slow in vitro, unless ATP is present. ATP causes a partial turnover of aminoacyl tRNA in vitro, with no detectable decrease in the overall aminoacylation level of tRNA, which remains close to 100%. tRNA in the particles turns over rapidly in vivo. Since the ester bond of aminoacyl tRNA is stabilized inside the 42-S particles, this turnover cannot be a consequence of spontaneous deacylation of tRNA, followed by reacylation by the aminoacyl-tRNA synthetases associated with the particles. We rather consider this turnover as reflecting a true metabolic activity of the particles, and a direct or indirect involvement of these particles in the oocyte's protein-synthesizing system.

Adenosine Triphosphate↗
PubMed1984

Structural features of yeast tRNA genes which affect transcription factor binding.

Transcription of yeast tRNA genes in vitro requires, in addition to RNA polymerase III, two accessory factors which are resolved by ion-exchange chromatography. One of these transcription factors (factor C) binds to tRNA genes. The stability of factor C-tRNA gene complexes is gene-dependent: the tRNAAGGArg gene forms a highly stable complex while tRNA3Leu and tRNATyr gene complexes are unstable under our standard assay conditions. To determine how differences in tRNA gene structure affect factor C binding, mutant tRNATyr genes, internally deleted tRNA3Leu genes and hybrid transcription units containing both tRNATyr and tRNA3Leu segments were compared in their abilities to stably bind factor C. Sequence changes in either of the two highly conserved promoter elements (A block and B block) affect factor C complex stability. Changes towards the consensus sequence increase complex stability while changes away from the consensus sequence drastically reduce stability. Also, the distance separating the A and B blocks affects complex stability; 34-53 bp gives highest stability. These results indicate that the stable binding of transcription factor C to tRNA genes involves interactions with both A block and B block sequences.

Base Sequence↗