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PubMedDate not supplied

[Conformational transition within the A-form of complementary nucleic acids in solution].

Circular dichroic spectra of A-DNA in 78% ethanol and of tRNA in water and ethanol solutions have been studied at different concentrations of NaCl. An increase in the Na+ concentration from 0.5.10(-4) M to 5.10(-4) M results in a shift of the positive CD band at 264 nm of the A-DNA to a longer wavelength, 272 nm. Simultaneously, the magnitude of the 210 nm band decreases. By contrast in the case of tRNA in water solution an increase in NaCl content results in straight opposite shifts of the CD spectra. This opposite behaviour is shown to the due to a difference in ions effects in water and water-ethanol solutions, since tRNA in the ethanol solution behaves in the same way as A-DNA does in 78% ethanol. We suppose that in aqueous solution in increase in the cation concentration would stabilize the helical conformations with progressively decreasing narrow groove, i. e. more wound. At a high concentration of ethanol (60--80%) the formation of specific complex between the hydrated cations and the double-stranded regions should be taken into consideration. Thus, the hydrated cations may insert into the deep groove exerting the opposite effect of unwinding.

Circular Dichroism↗
PubMed1979

The influence of ribonucleoside triphosphates, and other factors, on the formation of very-salt-stable RNA-polymerase . su+III-tRNA(tRNATyr)-promoter complexes.

The formation of a stable RNA-polymerase . su+III-tRNA-promoter complex was found to require sigma factor and the incorporation of ribonucleoside triphosphates which match the 5' sequence of the su+III tRNA transcript. This complex, stable to at least 2 M KCl, can be retained on a Millipore filter. Its formation closely parallels the extent of transcription obtained from the su+III tRNA promoter in response both to increasing ionic strength and to temperature during incubation of RNA polymerase with the DNA. The RNA-polymerase . DNA complex retained during this assay therefore appears to relate directly to that formed during promoter-directed transcription. The formation of RNA-polymerase . su+III-tRNA-promoter complexes is sensitive to the presence of ppGpp.

Base Sequence↗
PubMed1977

Dependence of mitochondrial protein synthesis initiation on formylation of the initiator methionyl-tRNAf.

The effect of N10-formyl-H4folate on mitochondrial peptide chain initiation has been studied in isolated mitochondria of Saccharomyces cerevisiae. The addition of N10-formyl-H4-folate strongly stimulates the incorporation of amino acids into mitochondrial protein at both 6 and 15 mm Mg2+. Still higher stimulation (up to 10-fold) has been obtained in the production of de novo synthesized initial peptides, measured as peptidyl puromycin derivatives. The maximum effect is observed at 0.1 mM N10-formyl-H4folate. At 5 mM puromycin, the ratio formylated/unformylated peptides is 3, as shown by electrophoretic analysis. At 10 mM puromycin, the ratio is increased to more than 6. This is due to the presence of deformylase and amidohydrolase activities, which are more effective the longer the initial peptide is synthesized; at increasing puromycin concentrations, progressively shorter peptide chains are formed. Chemically synthesized fMet-puromycin and Met-puromycin are virtually stable when incubated with intact or frozen and thawed mitochondria. More careful kinetic analysis shows an early cessation of the initial peptide formation in the samples without N10-formyl-H4-folate. This indicates that the formylation of methionyl-tRNA formylatable species is an absolute requirement for mitochondrial peptide chain initiation.

Drug Stability↗
PubMed2001

tRNA-guanine transglycosylase from Escherichia coli: molecular mechanism and role of aspartate 89.

The enzyme tRNA-guanine transglycosylase (TGT, EC 2.4.2.29) catalyzes a posttranscriptional transglycosylation reaction involved in the incorporation of the modified base queuine [Q, 7-(4,5-cis-dihydroxy-2-cyclopenten-1-ylaminomethyl)-7-deazaguanine] into tRNA. Previously, the crystal structure of the TGT from Zymomonas mobilis was solved in complex with preQ(1) (the substrate for the eubacterial TGT) [Romier et al. (1996) EMBO J. 15, 2850-2857]. An aspartate residue at position 102 (position 89 in the Escherichia coli TGT) was proposed to play a nucleophilic role in an associative catalytic mechanism. Although this is an attractive and precedented mechanism, a dissociative mechanism is equally plausible. In a dissociative mechanism, aspartate 89 would provide electrostatic stabilization of an oxocarbenium ion intermediate that is formed by dissociation of guanine. To clarify the nature of the catalytic mechanism of TGT, we have generated and characterized four mutations of aspartate 89 in the E. coli TGT (alanine, asparagine, cysteine, and glutamate). All four mutant TGTs were able to noncovalently bind tRNA, but only the glutamate mutant was able to form a stable complex with the RNA substrate under denaturing conditions that was comparable to wild type. Furthermore, the glutamate mutant was the only mutant TGT that demonstrated significant activity. Kinetic parameters were determined for this enzyme and shown to be comparable to wild type, revealing that the enzyme is considerably tolerant of the positioning of the carboxylate. Under conditions of high enzyme concentrations and long time courses, the alanine, asparagine, and cysteine mutants showed very low levels (ca. 10(3)-fold lower than wild type) of activity that were linear with respect to enzyme concentration and dependent upon pH in a fashion similar to that of the wild type. However, the observed initial velocities were too low to accurately determine k(cat) and K(m) values. We hypothesize that the activity observed for these mutants is most likely derived from host strain TGT (wt) contamination. These results are most consistent with aspartate 89 acting as a nucleophile in an associative catalytic mechanism.

Aspartic Acid↗
PubMed2000

Sequence analysis and modular organization of threonyl-tRNA synthetase from Thermus thermophilus and its interrelation with threonyl-tRNA synthetases of other origins.

The gene encoding threonyl-tRNA synthetase (Thr-tRNA synthetase) from the extreme thermophilic eubacterium Thermus thermophilus HB8 has been cloned and sequenced. The ORF encodes a polypeptide chain of 659 amino acids (Mr 75 550) that shares strong similarities with other Thr-tRNA synthetases. Comparative analysis with the three-dimensional structure of other subclass IIa synthetases shows it to be organized into four structural modules: two N-terminal modules specific to Thr-tRNA synthetases, a catalytic core and a C-terminal anticodon-binding module. Comparison with the three-dimensional structure of Escherichia coli Thr-tRNA synthetase in complex with tRNAThr enabled identification of the residues involved in substrate binding and catalytic activity. Analysis by atomic absorption spectrometry of the enzyme overexpressed in E. coli revealed the presence in each monomer of one tightly bound zinc atom, which is essential for activity. Despite strong similarites in modular organization, Thr-tRNA synthetases diverge from other subclass IIa synthetases on the basis of their N-terminal extensions. The eubacterial and eukaryotic enzymes possess a large extension folded into two structural domains, N1 and N2, that are not significantly similar to the shorter extension of the archaebacterial enzymes. Investigation of a truncated Thr-tRNA synthetase demonstrated that domain N1 is not essential for tRNA charging. Thr-tRNA synthetase from T. thermophilus is of the eubacterial type, in contrast to other synthetases from this organism, which exhibit archaebacterial characteristics. Alignments show conservation of part of domain N2 in the C-terminal moiety of Ala-tRNA synthetases. Analysis of the nucleotide sequence upstream from the ORF showed the absence of both any anticodon-like stem-loop structure and a loop containing sequences complementary to the anticodon and the CCA end of tRNAThr. This means that the expression of Thr-tRNA synthetase in T. thermophilus is not regulated by the translational and trancriptional mechanisms described for E. coli thrS and Bacillus subtilis thrS and thrZ. Here we discuss our results in the context of evolution of the threonylation systems and of the position of T. thermophilus in the phylogenic tree.

Amino Acid Motifs↗
PubMed1975

Effect of heat shock on the synthesis of low molecular weight RNAs in drosophilia: accumulation of a novel form of 5S RNA.

The synthesis and stability of low molecular weight RNAs following heat shock in Drosophilia melanogaster cell cultures have been examined. When cultures are raised from 25 degrees C to 37 degrees C, the synthesis of tRNA and at least two other low molecular weight RNAs continues at the 25 degree C rate. 5.8S ribosomal RNA and most of the low molecular weight nuclear RNAs are not synthesized. The synthesis of 5S ribosomal RNA is greatly reduced. A large amount of an RNA of about 135 nucleotides in length accumulates at 37 degrees C. Nucleotide sequence analysis reveals that this RNA is a novel form of 5S RNA with approximately 15 additional nucleotides at its 3' end.

Base Sequence↗
PubMed2003

Roles of 5-substituents of tRNA wobble uridines in the recognition of purine-ending codons.

Many tRNA molecules that recognize the purine-ending codons but not the pyrimidine-ending codons have a modified uridine at the wobble position, in which a methylene carbon is attached directly to position 5 of the uracil ring. Although several models have been proposed concerning the mechanism by which the 5-substituents regulate codon-reading properties of the tRNAs, none could explain recent results of the experiments utilizing well-characterized modification-deficient strains of Escherichia coli. Here, we first summarize previous studies on the codon-reading properties of tRNA molecules with a U derivative at the wobble position. Then, we propose a hypothetical mechanism of the reading of the G-ending codons by such tRNA molecules that could explain the experimental results. The hypothesis supposes unconventional base pairs between a protonated form of the modified uridines and the G at the third position of the codon stabilized by two direct hydrogen bonds between the bases. The hypothesis also addresses differences between the prokaryotic and eukaryotic decoding systems.

Anticodon↗
PubMed1997

Structure and aminoacylation capacities of tRNA transcripts containing deoxyribonucleotides.

The contribution of the ribose 2'-hydroxyls to RNA structure and function has been analyzed, but still remains controversial. In this work, we report the use of a mutant T7 RNA polymerase as a tool in RNA studies, applied to the aspartate and methionine tRNA aminoacylation systems from yeast. Our approach consists of determining the effect of substituting natural ribonucleotides by deoxyribonucleotides in RNA and, thereby, defining the subset of important 2'-hydroxyl groups. We show that deoxyribose-containing RNA can be folded in a global conformation similar to that of natural RNA. Melting curves of tRNAs, obtained by temperature-gradient gel electrophoresis, indicate that in deoxyribo-containing molecules, the thermal stability of the tertiary network drops down, whereas the stability of the secondary structure remains unaltered. Nuclease footprinting reveals a significant increase in the accessibility of both single- and double-stranded regions. As to the functionality of the deoxyribose-containing tRNAs, their in vitro aminoacylation efficiency indicates striking differential effects depending upon the nature of the substituted ribonucleotides. Strongest decrease in charging occurs for yeast initiator tRNA(Met) transcripts containing dG or dC residues and for yeast tRNA(Asp) transcripts with dU or dG. In the aspartate system, the decreased aminoacylation capacities can be correlated with the substitution of the ribose moieties of U11 and G27, disrupting two hydrogen bond contacts with the synthetase. Altogether, this suggests that specific 2'-hydroxyl groups in tRNAs can act as determinants specifying aminoacylation identity.

Base Sequence↗