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Biomedical subjects

M Sprinzl

Publications and source records attributed to M Sprinzl.

At least 109 records · Page 6Linked to original sources
PubMed1986

Fluorescence labeling of an aminoacyl-tRNA at the 3'-end and its interaction with elongation factor Tu.GTP.

A new approach for the fluorescence labeling of an aminoacyl-tRNA at the 3'-end is applied to study its interaction with bacterial elongation factor Tu (EF-Tu) and GTP at equilibrium. The penultimate cytidine residue in yeast tRNATyr-C-C-A was replaced by 2-thiocytidine (s2C). The resulting tRNATyr-C-s2C-A was aminoacylated and then alkylated at the s2C residue with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (1,5-I-AEDANS). A greater than 100% increase in the intensity of fluorescence emission of the modified Tyr-tRNATyr-C-s2C(AEDANS)-A was observed upon interaction with EF-Tu.GTP. A ternary complex dissociation constant of 1.27 X 10(-8) M was calculated from this direct interaction. Using such fluorescent aminoacyl-tRNA, the affinity of any unmodified aminoacyl-tRNA can be determined by competition experiments. By this approach, we show here that the affinity of unmodified Tyr-tRNATyr-C-C-A is identical to that of the modified Tyr-tRNATyr. This indicates that the fluorescence labeling procedure applied does not alter the affinity of the aminoacyl-tRNA for EF-Tu.GTP. The introduction of 2-thiocytidine into nucleic acids and their labeling with spectroscopic reporter groups may provide a unique means of investigating various types of nucleic acid-protein interactions.

Cytidine↗
PubMed1986

Transfer ribonucleic acid populations in concanavalin-A-stimulated bovine lymphocytes.

Transfer RNA isolated from lymphocytes stimulated by concanavalin A and that from resting cells were compared with respect to amino-acid acceptance, integrity of the CCA-terminus, extent of modification and isoacceptor distribution. Following growth stimulation the overall amino-acid acceptance of the tRNA is elevated, in particular the relative acceptor activities for threonine and arginine are increased. The reduced acceptor activity of the tRNA from the quiescent cells is not due to a preferential degradation of the CCA-end, since it persists even in the presence of ATP(CTP):tRNA nucleotidyltransferase. We therefore conclude that this reduced activity is caused by structural differences of the tRNAs. The content of modified nucleotides in newly synthesized tRNA from lymphocytes cultured in the presence and absence of concanavalin A was determined. tRNA from resting cells was found to be undermodified with respect to pseudouridine and dihydrouridine. Upon monitoring the tRNA isoacceptor distribution by affinity chromatography on immobilized elongation factor Tu and subsequent two-dimensional gel electrophoresis, a preferential synthesis of particular lysine- and threonine-accepting tRNAs was observed upon mitogenic stimulation. Evidently, a specific tRNA population is needed by the proliferating cells. These results are discussed in view of the hypothesis that the commitment of lymphocytes to proliferation is at least in part under translational control.

Animals↗
PubMed1986

Spin-labelled analogues of GDP and GTP as site-specific reporter groups for guanosine nucleotide-binding proteins.

New derivatives of GDP and GTP have been synthesized for the spectroscopic investigation of the interaction between guanosine nucleotides and guanosine nucleotide-binding proteins. The 3'-hydroxyl group in these nucleotides was replaced by a 3'-amino group, which was further derivatized by the introduction of a spin-label reporter group. The biological activity of 3'SL-GDP and 3'SL-GTP could be demonstrated by measuring the interaction of these spin-labelled derivatives with bacterial elongation factor Tu. The amino modification and spin labelling only slightly influenced the affinity of the guanosine nucleotides for EF-Tu from Escherichia coli or Thermus thermophilus. Electron paramagnetic resonance (EPR) measurements revealed a strong immobilization of the labelled nucleotides upon binding to T. thermophilus EF-Tu. Significant differences between the spectra of EF-Tu X 3'SL-GDP, EF-Tu X 3'SL-GTP and aminoacyl-tRNA X EF-Tu X 3'SL-GTP ternary complexes were observed. Our data demonstrate that spin-labelled guanosine nucleotides can be used as sensitive spectroscopic probes for the investigation of the local environment of the nucleotide-binding site during distinct functional states of a guanosine nucleotide-binding protein.

Chromatography, Gel↗
PubMed1986

Ser-tRNAs from bovine mitochondrion form ternary complexes with bacterial elongation factor Tu and GTP.

Transfer ribonucleic acids were isolated from mitochondria of bovine heart and aminoacylated in vitro by a crude mitochondrial enzyme. Ser-tRNASerUCN and Ser-tRNASerAGY were isolated and characterized by partial sequencing. Although these tRNAs possess unique structural features not found in any bacterial tRNA, they form a ternary complex with elongation factor from the extreme thermophilic bacterium Thermus thermophilus and GTP.

Amino Acids↗
PubMed1985

Interaction of methionine-specific tRNAs from Escherichia coli with immobilized elongation factor Tu.

The interaction of three different Met-tRNAsMet from E. coli with bacterial elongation factor (EF) Tu X GTP was investigated by affinity chromatography. Met-tRNAfMet which lacks the base pair at the end of the acceptor stem binds only weakly to EF-Tu X GTP, while Met-tRNAmMet has a high affinity for the elongation factor. A modified Met-tRNAfMet which has a C1-G72 base pair binds much more strongly to immobilized EF-Tu X GTP than the native aminoacyl(aa)-tRNA with non-base-paired C1A72 at this position, demonstrating that the base pair including the first nucleotide in the tRNA is one of the essential structural requirements for the aa-tRNA X EF-Tu X GTP ternary complex formation.

Chromatography, Affinity↗
PubMed1985

Analysis of modification-dependent structural alterations in the anticodon loop of Escherichia coli tRNAArg and their effects on the translation of MS2 RNA.

The conformation of the anticodon loop of Escherichia coli tRNAArg was investigated. It is shown that the structure of the anticodon loop is influenced by the base composition of the anticodon stem, and the natural modification of the nucleoside residue 32 in the anticodon loop. The structural effects detected by analysis of the accessibility of the anticodon loop to nuclease S1 could be correlated with the ability of different Arg-tRNAArg species to suppress frame-shifting during translation of MS2 RNA.

Anticodon↗
PubMed1985

Interaction of unfolded tRNA with the 3'-terminal region of E. coli 16S ribosomal RNA.

Fragments of tRNA possessing a free TpsiC-loop or a free D-loop form stable complexes with the colicin fragment (1494-1542) of 16S ribosomal RNA from E. coli. The colicin fragment does not bind to tRNA in which the T-loop and the D-loop are involved in tertiary interactions. Colicin cleavage of the 16S rRNA from E. coli is inhibited by aminoacyl-tRNA or tRNA fragments, indicating that a similar interaction may take place on the intact 70S ribosomes. The oligonucleotide d(G-T-T-C-G-A)homologous to the conserved sequence G-T-psi-C-Pu-(m1)A in the TpsiC-region of many elongator tRNAs binds to the conserved sequence U-C-G-mU-A-A-C (1495-1501) of the 16S rRNA. It is suggested that the 3'-end of the 16S rRNA may provide the part of the binding site for the elongator tRNAs on bacterial ribosomes.

Colicins↗
PubMed1985

Synthesis and characterization of poly[d(G-z5C)]. B-Z transition and inhibition of DNA methylase.

Deoxy-5-azacytidine 5'-triphosphate was synthesized and used as a substrate for the enzymatic synthesis of the polynucleotide poly[d(G-z5C)]. Whereas the triphosphate decomposes in solution, the azacytosine analogue incorporated into DNA is stable under conditions preserving the double-helical structure. Poly[d(G-z5C)] undergoes the transition to the left-handed Z conformation at salt (NaCl and MgCl2) concentrations approximately 30% higher than those required for unsubstituted poly[d(G-C)]. However, the incorporation of azacytidine potentiates the formation at room temperature of the Z helix stabilized by the transition metal Mn2+; in the case of poly[d(G-C)], a heating step is required. The spectral properties of the two polymers in the B and Z forms are similar. Both left-handed forms are recognized by anti-Z DNA immunoglobulins, indicating that the DNAs bear common antigenic features. Poly[d(G-z5C)] is not a substrate for the DNA cytosine 5-methyltransferase from human placenta. It is a potent inhibitor of the enzyme when tested in a competitive binding assay. These results are compatible with a very strong, possibly covalent, mode of interaction between methyltransferases and DNA containing 5-azacytosine.

Azacitidine↗
PubMed1985

Serine-specific tRNAs in Escherichia coli: relative abundance and sequence.

Serine isoaccepting tRNAs were isolated from bulk tRNA of Escherichia coli by affinity chromatography on immobilized bacterial elongation factor Tu and their relative abundance was determined. The three major species, which are sufficient to read all six serine codons, were identified by sequencing. The sequence of a novel tRNASer with the anticodon GGA was elucidated.

Base Sequence↗
PubMed1984

Aminoacyl RNA domain of turnip yellow mosaic virus Val-RNA interacting with elongation factor Tu.

Turnip yellow mosaic virus (TYMV) Val-RNA forms a complex with the peptide elongation factor Tu (EF-Tu) in the presence of GTP: the Val-RNA is protected by EF-Tu.GTP from non-enzymatic deacylation and nuclease digestion. The determination of the length of the shortest TYMV Val-RNA fragment that binds EF-Tu.GTP leads us to conclude that the valylated aminoacyl RNA domain equivalent in tRNAs to the continuous helix formed by the acceptor stem and the T arm is sufficient for complex formation. Since the aminoacyl RNA domain is also sufficient for adenylation by the ATP(CTP):tRNA nucleotidyltransferase, an analogy can be drawn between these two tRNA-specific proteins.

Journal Article↗
PubMed1984

Chemical synthesis of 5-azacytidine nucleotides and preparation of tRNAs containing 5-azacytidine in its 3'-terminus.

5-azacytidine-5'-triphosphate prepared from 5-azacytidine by chemical phosphorylation is a substrate for AMP (CMP) tRNA nucleotidyl transferase from yeast. tRNAsPhe from yeast containing 5-azacytidine in their 3'-termini were prepared enzymatically. tRNAPhe-Cpn5CpA and tRNAPhe-n5Cpn5CpA can be aminoacylated by phenylalanyl-tRNA synthetase from yeast and they are active in the poly(U)-dependent synthesis of poly(Phe) on E. coli ribosomes. The decomposition of 5-azacytidine via hydrolysis of the triazine ring is significantly accelerated by a phosphate group on the 5'-position of the nucleotide. After the incorporation of 5-azacytidine-5'-phosphate into a polynucleotide chain the rate of hydrolysis of the triazine ring decreases considerably.

Azacitidine↗
PubMed1984

Isolation of tRNA isoacceptors by affinity chromatography on immobilized bacterial elongation factor Tu.

Elongation factor Tu from Escherichia coli or Thermus thermophilus was immobilized on cyanogen bromide-activated Sepharose. Immobilized elongation factor Tu X GDP could be converted to Tu X GTP, which is able to bind aminoacyl-tRNA. When bulk tRNA from E. coli, baker's yeast, or bovine liver was aminoacylated by one amino acid only, the resulting aminoacyl-tRNA could be separated in one step from the rest of the tRNA using an affinity column of immobilized elongation factor Tu X GTP. Specific tRNA isoacceptors can be isolated in amounts sufficient for gel electrophorectic analysis, sequence determinations, and hybridization experiments.

Amino Acids↗