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At least 775 records · Page 43Linked to original sources
PubMed1984

Difference in radiosensitivity of valyl-tRNA synthetases isolated from chick embryo liver and brain.

The sensitivity in vitro against gamma-radiation of valyl-tRNA synthetase (VRS) from chick embryo brain proved to be higher than that of liver VRS. In order to study this phenomenon properties of VRS isolated from both organs were compared. Both enzymes have the same molecular weight (Mr = 110 000) determined by equilibrium sedimentation and by gel filtration. Electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate showed no evidence for subunit structure. The optimal reaction conditions in valyl-tRNA formation were found to be the same, except the pH values. Km values of the reactions were also similar. Both enzymes reacted with the tRNAs isolated from the other organ. The stability during storage of the liver VRS was higher than that of the brain VRS. In the brain VRS molecule one buried and eleven free SH groups could be detected and divided into three classes with different reactivities. In 1 mol of liver VRS one buried and nine free thiol groups were determined and these could be divided only into two classes. These observations suggest the existence of two VRS isoenzymes with different radiosensitivities: the more sensitive occurs mainly in brain and the less sensitive mainly in the liver of chick embryo.

Amino Acyl-tRNA Synthetases↗
PubMed1998

C-terminal truncation of yeast SerRS is toxic for Saccharomyces cerevisiae due to altered mechanism of substrate recognition.

Like all other eukaryal cytosolic seryl-tRNA synthetase (SerRS) enzymes, Saccharomyces cerevisiae SerRS contains a C-terminal extension not found in the enzymes of eubacterial and archaeal origin. Overexpression of C-terminally truncated SerRS lacking the 20-amino acid appended domain (SerRSC20) is toxic to S. cerevisiae possibly because of altered substrate recognition. Compared to wild-type SerRS the truncated enzyme displays impaired tRNA-dependent serine recognition and is less stable. This suggests that the C-terminal peptide is important for the formation or maintenance of the enzyme structure optimal for substrate binding and catalysis.

Amino Acid Sequence↗
PubMed1976

Preparation and characterization of eukaryotic initiation factor EIF-3. Formation of binary (EIF-3-Met-tRNAf) and ternary (EIF-3-Met-tRNAf-GTP) complexes.

The 133,000 X g supernatant fraction prepared from ascites cells in 20 mM KCl (low CKl supernatant) contained the initiation factors EIF-1 and EIF-2 (and the elongation factore EF-1 and EF-2) but lacked EIF-3; thus, low KCl supernatant could be used to assay for EIF-3. EIF-3 was prepared from a crude initiation factor perparation (a 250 mM KCl extract of ascites cell ribosomes precipitated with 70% saturated ammonium sulfate) by chromatography on DEAE-Sephadex A-50 and hydroxylapatite. The EIF-O had no detectable EIF-1 and little or no EIF-2. Factor EIF-3 was required fro translation of encephalomyocarditis virus RNA. The molecular weight of EIF-3 was estimated by Sephadex G-200 filtration to be 139,000; the sedimentation coefficient was calculated to be about 5.8. EIF-3 formed a binary complex specifically with the initiator tRNA, Met-tRNAf, and if GTP was present the factor formed a ternary complex (EIF-3-Met-tRNAf-GTP). The EIF-3 preparation had no methionyl-tRNA synthetase activity to account for binding. Complex-formation was with eukaryotic Met-tRNAf and no other aminoacyl-tRNA. The binary and ternary complexes were retained quantitatively on Millipore filters (which was the most convenient assay), but they could also be demonstrated by filtration through Sephadex G-100 or by glycerol gradient centrifugation. GTP increased the rate, the amount, and the stability of complex formed; the ration of GTP to Met-tRNAf in the ternary complex appeared to be 1. The binary and the ternary complexes transferred Met-tRNAf to the 40 S ribosomal subunits, but not to 60 S subparticles. The factor-dependent binding of Met-tRNAf to the 40 S subunit did not require mRNA (or GTP). In the presence of 60 S subunits, the initiator tRNA bound to 40 S subunits was not transferred to 80 S ribosomes even if mRNA was added--that reaction may require another initiation factor. Treatment of EIF-3 with N-ethylmaleimide led to loss of its activity in complex formation and in support of the translation of encephalomyocarditis virus RNA. In addition to forming the binary and ternary complexes, and supporting the translation of encephalomyocarditis virus RNA, EIF-3 also increases the number of free ribosomal subunits by either preventing their association or causing dissociation of 80 S couples.

Animals↗
PubMed1995

Modified nucleoside-dependent transition metal binding to DNA analogs of the tRNA anticodon stem/loop domain.

Biologically active DNA analogs of tRNAPhe (tDNAPhe) were used to investigate metal ion interaction with tRNA-like structures lacking the 2'OH. Binding of Mg2+ to the 76 oligonucleotide tDNAPhe, monitored by circular dichroism spectroscopy, increased base stacking and thus the conformational stability of the molecule. Mg2+ binding was dependent on a d(m5C) in the anticodon region. In contrast to Mg2+, Cd2+ decreased base stacking interactions, thereby destabilizing the molecule. Since alterations in the anticodon region contributed to most of the spectral changes observed, detailed studies were conducted with anticodon hairpin heptadecamers (tDNAPheAC). The conformation of tDNAPheAC-d(m5C) in the presence of 1 mM Cd2+, Co2+, Cr2+, Cu2+, Ni2+, Pb2+, VO2+ or Zn2+ differed significantly from that of the biologically active structure resulting from interaction with Mg2+, Mn2+ or Ca2+. Nanomolar concentrations of the transition metals were sufficient to denature the tDNAPheAC-d(m5C) structure without catalyzing cleavage of the oligonucleotide. In the absence of Mg2+ and at [Cd2+] to [tDNAPheAC-d(m5C)] ratios of approximately 0.2-1.0, tDNAPheAC-d(m5C40) formed a stable conformation with one Cd2+ bound with a Kd = 3.7 x 10(-7) M. In contrast to Mg2+, Cd2+ altered the DNA analogs without discriminating between modified and unmodified tDNAPheAC. This ability of transition metals to disrupt higher order DNA structures, and possibly RNA, at microM concentrations, in vitro, demonstrates that these structures are potential targets in chronic metal exposure, in vivo.

Anticodon↗
PubMed2002

Functional evidence for D- and T-loop interactions in tmRNA.

During bacterial protein synthesis, stalled ribosomes can be rescued by tmRNA, a molecule with both tRNA and mRNA features. The tRNA region of tmRNA has sequence similarity with tRNA(Ala) and also has a clover-leaf structure folded similarly as in canonical tRNAs. Here we propose the L-shape of tmRNA to be stabilized by two tertiary interactions between its D- and T-loop on the basis of phylogenetic and experimental evidence. Mutational analysis clearly demonstrates a tertiary interaction between G(13) and U(342). Strikingly, this in evolution conserved interaction is not primarily important for tmRNA alanylation and for binding to elongation factor Tu, but especially for a proper functioning of SmpB.

Alanine↗
PubMed1999

A novel method for increasing the transformation efficiency of Escherichia coli-application forbacterial artificial chromosome library construction.

Bacterial artificial chromosome (BAC) libraries play a pivotal role in genomics studies. A crucial step in BAC library construction is the transformation of Escherichia coli by electroporation. Absolute efficiency (cfu/microgram DNA) is affected by a number of factors including the topological form and treatment of DNA samples. Here we report a simple new protocol using tRNA assisted precipitation that increased transformation efficiency by 70-fold for BAC ligations and up to 400-fold for plasmid ligations. The mechanism may involve altering or stabilizing the topographical form of the DNA molecules.

Chromosomes, Bacterial↗
PubMed1994

Escherichia coli elongation-factor-Tu mutants with decreased affinity for aminoacyl-tRNA.

The two evolutionary well-conserved histidine residues, His66 and His118, of Escherichia coli elongation factor Tu have been subjected to mutational analysis. The two histidines have each been replaced by alanines, denoted H66A and H118A, respectively. His118 has also been substituted by glutamate, H118E. The three mutants have been characterized with respect to thermostability, GTPase activity and affinity for aminoacylated tRNA. Most conspicuously, the tRNA affinity is reduced or almost abolished. k-1 for dissociation of the ternary complex increases by factors of 14, 40 and 48 for H66A, H118A and H118E, respectively, when compared to the wild type. The half-lives for the non-enzymic deacylation of aminoacylated tRNA in the ternary complex are 391, 107, 69, 54 and 61 min for wild type, H66A, H118A, H118E and free aminoacylated tRNA, respectively. The Kd is about 20-times higher for H66A compared to wild type. Our results strongly suggest that His66 and His118 play major roles in stabilization of the ternary complex.

Base Sequence↗
PubMed2003

Glutamate counteracts the denaturing effect of urea through its effect on the denatured state.

The urea induced equilibrium denaturation behavior of glutaminyl-tRNA synthetase from Escherichia coli (GlnRS) in 0.25 m potassium l-glutamate, a naturally occurring osmolyte in E. coli, has been studied. Both the native to molten globule and molten globule to unfolded state transitions are shifted significantly toward higher urea concentrations in the presence of l-glutamate, suggesting that l-glutamate has the ability to counteract the denaturing effect of urea. d-Glutamate has a similar effect on the equilibrium denaturation of glutaminyl-tRNA synthetase, indicating that the effect of l-glutamate may not be due to substrate-like binding to the native state. The activation energy of unfolding is not significantly affected in the presence of 0.25 m potassium l-glutamate, indicating that the native state is not preferentially stabilized by the osmolyte. Dramatic increase of coefficient of urea concentration dependence (m) values of both the transitions in the presence of glutamate suggests destabilization and increased solvent exposure of the denatured states. Four other osmolytes, sorbitol, trimethylamine oxide, inositol, and triethylene glycol, show either a modest effect or no effect on native to molten globule transition of glutaminyl-tRNA synthetase. However, glycine betaine significantly shifts the transition to higher urea concentrations. The effect of these osmolytes on other proteins is mixed. For example, glycine betaine counteracts urea denaturation of tubulin but promotes denaturation of S228N lambda-repressor and carbonic anhydrase. Osmolyte counteraction of urea denaturation depends on osmolyte-protein pair.

Amino Acyl-tRNA Synthetases↗
PubMed2000

Immobilized Tryptophanyl-tRNA Synthetase from Bacillus subtilis.

In order to investigate the recognition mechanism and the relationship between structure and function of tRNA(Trp) with tryptophanyl-tRNA synthetase (TrpRS), TrpRS from Bacillus subtilis was purified and immobilized on CNBr-activated Sepharose 4B. Protein recovery and activity recovery of the immobilization were 95.5% and 31.3%, respectively. Properties of immobilized TrpRS were studied in detail. The thermal stability and the shelf stability of immobilized TrpRS were much higher than those of the native TrpRS. Besides these, the immobilized TrpRS, with good operation stability, had increased optimum temperature and optimum pH. A 56-base single-stranded RNA library containing 20 consecutive completely randomized bases was subjected to 3 successive rounds of immobilized TrpRS column selection with SELEX method, resulting in a sharp increase of the percentage of the RNA pool that could bind immobilized TrpRS from 4.3% for the first round pool to 14.7% for the third-round pool. After sequencing the third-round RNA pool, a RNA secondary structure resembling the structure of the acceptor stem in tRNA(Trp) was obtained though the selection. All the results indicated that immobilized TrpRS could be used as an affinity chromatography matrix and was qualified for the SELEX of a RNA pool simulating tRNA(Trp) molecule.

Journal Article↗
PubMed1978

Methionyl-tRNA synthetase from Escherichia coli. Absence of interaction between the metal ion and the purine ring of ATP in the L-methionine activation site.

Enhancement of the nuclear relaxation rates by manganese has been used to derive manganese--purine-ring distances in the activation site of methionyl-tRNA synthetase. This is possible with the help of an abortive complex between the enzyme, methionine, adenosine, pyrophosphate and manganese which simulates an intermediate species of the activation reaction. It is found that the distances between the manganese ion and the purine ring are too high (greater than 0.8 nm) to allow interaction between them. Thus, metal-purine interaction is involved neither in the catalytic mechanism nor in the stabilization of abortive synergistic complexes [S. Blanquet, G. Fayat and J. P. Waller (1975) J. Mol. Biol. 94, 1-15].

Adenosine↗
PubMed1987

Unrelated leader sequences can efficiently promote human tRNA gene transcription.

The 5'-leader sequence of a human tRNA gene encoding the major tRNA(IACVa 1) species was replaced by several unrelated sequences of human and bacterial origin. Transcription in a HeLa cell extract revealed an extragenic control region (ECR) between positions -51 and -16. Competition assays demonstrate that the wild-type ECR acts as a positive modulator of transcription factor binding. The amount of active transcription complex formed is shown to be dependent on the ECR, whereas the stability of transcription complexes formed under the control of wild-type and mutant ECRs seems not to be affected. One bacterial DNA provided transcription controlling properties indistinguishable from those of the natural human leader sequence. The poor homology between these two sequences indicates that ECRs of human tRNA genes do not consist of highly conserved boxes like intragenic control regions, but of fairly individual DNA elements.

Base Sequence↗
PubMed2003

Feedback regulation of MAPK signalling by an RNA-binding protein.

Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes that convert extracellular signals into various outputs such as cell growth, differentiation and cell death. MAPK phosphatases selectively inactivate MAPKs by dephosphorylating critical phosphothreonine and phosphotyrosine residues. The transcriptional induction of MAPK phosphatase expression by various stimuli, including MAPK activation, has been well documented as a negative-feedback mechanism of MAPK signalling. Here we show that Rnc1, a novel K-homology-type RNA-binding protein in fission yeast, binds and stabilizes Pmp1 messenger RNA, the MAPK phosphatase for Pmk1 (refs 10, 11). Rnc1 therefore acts as a negative regulator of Pmk1 signalling. Notably, Pmk1 phosphorylates Rnc1, causing enhancement of the RNA-binding activity of Rnc1. Thus, Rnc1 is a component of a new negative-feedback loop that regulates the Pmk1 pathway through its binding to Pmp1 mRNA. Our findings--the post-transcriptional mRNA stabilization of a MAPK phosphatase mediated by an RNA-binding protein--provide an additional regulatory mechanism for fine-tuning of MAPK signalling pathways.

Deoxyribonucleases↗
PubMed1980

Some aspects of chemical synthesis of oligoribonucleotides.

A simple synthesis of ribothymidine 3'-phosphate from uridine 3'-phosphate was described. Crystalline 5'-O-trityl- or 5'-O-monomethoxytrityl-derivatives of ribonucleotides are easily prepared and purified in relatively large batches through choices of appropriate solvents. Acylimidazoles together with a strong nitrogeneous base have been successfully used for the acylation of the hydroxyl and amino groups of the nucleoside and nucleotide, and the reaction mechanism was studied. The relative stability of different blocking groups of guanosine 3'-phosphate was compared. Oligoribonucleotide fragments including an octaribonucleotide of the T psi C- and D-loop of yeast alanine tRNA were synthesized. Quantitative analyses of the common ribo-nucleosides and -nucleotides were done by a modified RPC method.

Base Sequence↗