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At least 19 recordsLinked to original sources

PubMed1976

Status of tRNA charging, trinucleotide acceptor sequence and tRNA nucleotidyltransferase activity in the human placenta.

Samples of tRNA isolated from the cell sap of full-term human placenta were found to have a low capacity for accepting amino acids in the presence of partially purified synthetase preparations made from placental or rat liver cell sap. Gel electrophoresis of placental tRNA showed that part of this could be accounted for by gross degradation. The proportion of chargeable tRNA carrying amino acids was estimated by periodate oxidation followed by stripping and then charging with labeled amino acids. Only 50% of chargeable placental tRNA was in the charged state when isolated, whereas 87% of freshly isolated rat liver tRNA was found to be charged with amino acids. A fraction from placental cell sap was shown to have tRNA nucleotidyltransferase activity. When placental tRNA was incubated with this fraction and [3H]ATP or [3H]CTP, ATP was incorporated into about 12% of the tRNA molecules and CTP into 5-7%. When rat liver tRNA was used in place of placental tRNA, [3H]ATP was incorporated into less than 5% of the tRNA molecules. By using snake-venom diesterase over short periods of incubation, it was confirmed that the ATP had been incorporated terminally as AMP into the placental tRNA. These observations show that, in contrast to rat liver tRNA, tRNA prepared from human placenta is poorly charged with amino acids, many of the molecules lack the acceptor trinucleotide and there is extensive degradation beyond this stage.

Amino Acyl-tRNA Synthetases↗

PubMedDate not supplied

[Specific modification of phenylalanine:tRNA-ligases of E. coli MRE-600 with N-chlorambucilyl-14c-phenylalanyl-tRNA].

N-Chlorambucilyl-[14C]phenylalanyl-tRNA was used for the affinity modification of phenylalanine : tRNA-ligase from E. coli MRE-600. It has been found that N-chlorambucilyl-[14C]phenylalanyl-tRNA selectively inactivates phenylalanine : tRNA-lagase that results in formation of a covalent bond between the tRNA derivative and the enzyme at pH 5.8, 25 degrees C. The rate fall of the aminoacylation of tRNA with [14C]phenylalanine was observed after the enzyme incubation with N-chlorambucilyl-[14C]phenylalanyl-tRNA at pH 7.5, 25 degrees C. It has been shown that this modification results in a similar rate decrease of tRNA aminoacylation with [14C]phenylalanine, ATP-[32P]pyrophosphate exchange and reaction of the enzymatic deacylation of [14C]phenylalanyl-tRNA. This fact evidences in favour of the possibility of the alkylation to proceed in the proximity of the active centre of the enzyme. The covalent complex obtained seems to be an interesting model for the studies of the mechanisms involved in tRNA aminoacylation as well as for elucidation of the tertiary structure of tRNA bound with the enzyme.

Adenosine Triphosphate↗

PubMed1979

Immunochemical studies of beef pancreas tryptophanyl-tRNA synthetase and its fragments. Determination of the number of antigenic determinants and a comparison with tryptophanyl- tRNA synthetases from other sources and with reverse transcriptase from avian myeloblastosis virus.

The immunoglobulin G (IgG) fraction of the antiserum from rabbits immunized with homogeneous beef pancreas tryptophanyl-tRNA synthetase inhibits the enzyme activity in the reactions of both tRNATrp aminoacylation and tryptophan activation. Fab fragments of IgG act in a similar way. Common antigenic determinants have been detected in tryptophanyl-tRNA synthetases from beef, pig, chicken and rat livers using pure antibodies against beef pancreas tryptophanyl-tRNA synthetase. This observation indicates the evolutional stability of certain structural features of tryptophanyl-tRNA synthetases. The interaction of antibodies with the fragments of beef tryptophanyl-tRNA synthetase produced by endogenous and tryptic proteolysis of the enzyme has been studied. On third of the antiserum antibodies interacting with the C-terminal fragment of the enzyme (Mr approximately equal to 40000) inhibits its activity whereas the antibodies to the N-terminal fragment (Mr approximately equal to 20000) have no effect on the enzyme activity. The immunochemical identity of the two synthetase fragments differing in their enzymatic activity supports the assumption that the loss of enzymatic activity of the tryptic fragment is caused by lack of a small peptide which is retained in case of endogenous proteolysis; probably the amino acid residues of this peptide participate in formation of active centre of tryptophanyl-tRNA synthetase. A radioimmunochemical method is described for determining the number of antigenic determinants. One molecule of tryptophanyl-tRNA synthetase was found to bind 9 (+/- 1) molecules of Fab fragments. Antibodies against tryptophanyl-tRNA snythetase from beef pancreas do not inhibit noticeably the activity of reverse transcriptase from avian myeloblastosis virus. No antigenic determinants in common have been detected in reverse transcriptase and tryptophanyl-tRNA synthetase by radioimmunochemical assays.

Amino Acyl-tRNA Synthetases↗

PubMed1976

Distinct steps in the specific binding of tRNA to aminoacyl-tRNA synthetase. Temperature-jump studies on the serine-specific system from yeast and the tyrosine-specific system from Escherichia coli.

The kinetics of the interaction of tRNASer and seryl-tRNA synthetase from yeast as well as of tRNATyr and tyrosyl-tRNA synthetase from Escherichia coli have been investigated by temperature-jump experiments. It could be shown that complex formation proceeds in two distinct steps. This was demonstrated for both the first and the second binding site. The two-step mechanism was deduced from the characteristic concentration dependence of the relaxation times. Seryl-tRNA synthetase recombines with the first tRNA to form an intermediate complex (kI12, kI21), which is transformed in a fast reaction to the final 1:1 complex (kI23, kI32). At pH 7.2 with 0.1 M KCl the rate constants are: kI12 = 2.7 X 10(8) M-1 S-1; kI23, kI32). At pH 7.2 with 0.1 M KCl the rate constants are: kI12 = 2.7 x 10(8) M-1 S-1; kI21 = 220 S-1; kI23 = 760 S-1; kI32 = 330 S-1. The 1:1 complex can bind a second tRNA. At pH 7.2 without added salt the rate constants are: KII2 = 0.9 X 10(8) M-1 S-1; kII21 = 270 S-1; kII23 = 120 S-1; kII32 = 1250 S-1. The tyrosine-specific system behaves very similarly to the serine-specific system. Data are given for pH 7.2 (pH 6.0) for the binding of the second tRNA: kII12 = 1 X 10(8) (2.5 X 10(8)) M-1 S-1; kII21 = 470 (170) S-1; kII23 = 150 (530) S-1; kII32 = 1540 (720) S-1. The kinetic results are discussed in terms of their relevance to the recognition process and their relation to the anticooperative binding behaviour of tRNA to synthetase.

Amino Acyl-tRNA Synthetases↗

PubMed2023

Structural basis for pre-tRNA recognition and processing by the human tRNA splicing endonuclease complex.

Throughout bacteria, archaea and eukarya, certain tRNA transcripts contain introns. Pre-tRNAs with introns require splicing to form the mature anticodon stem loop. In eukaryotes, tRNA splicing is initiated by the heterotetrameric tRNA splicing endonuclease (TSEN) complex. All TSEN subunits are essential, and mutations within the complex are associated with a family of neurodevelopmental disorders known as pontocerebellar hypoplasia (PCH). Here, we report cryo-electron microscopy structures of the human TSEN-pre-tRNA complex. These structures reveal the overall architecture of the complex and the extensive tRNA binding interfaces. The structures share homology with archaeal TSENs but contain additional features important for pre-tRNA recognition. The TSEN54 subunit functions as a pivotal scaffold for the pre-tRNA and the two endonuclease subunits. Finally, the TSEN structures enable visualization of the molecular environments of PCH-causing missense mutations, providing insight into the mechanism of pre-tRNA splicing and PCH.

Humans↗

PubMed1979

Complete inactivation and labeling of methionyl-tRNA synthetase by periodate-treated initiator tRNA in the presence of sodium cyanohydridoborate.

Methionyl-tRNA synthetase from Escherichia coli can react with periodate-treated tRNA to form a Schiff's base through the epsilon-amino group of a lysine within the enzymic active center and the 2',3'-aldehyde groups created at the 3'-terminal ribose of tRNA. At alkaline pH, the Schiff's base equilibrium can be continuously and specifically displaced by reduction in situ with sodium cyanohydridoborate, which on the other hand leaves intact the reacting aldehyde groups of oxidized tRNA. The effects of temperature, pH and of reducing agent concentration on the rate and extent of reduction of the Schiff's base are analysed. Conditions are described (37 degrees C, pH 8.0, in the presence of 1 mM cyanohydridoborate) which allowed rapid and complete conversion of the monomeric trypsin-modified methionyl-tRNA synthetase into its 1:1 covalent complex with tRNAfMet.

Amino Acyl-tRNA Synthetases↗

PubMed1976

Subcellular localization of S-adenosyl-L-methionine:tRNA methyltransferases with aminoacyl-tRNA synthetases in human and mouse: normal and leukemic leukocytes.

The subcellular distributions of S-adenosyl-L-methionine:tRNA methyltransferases and aminoacyl-tRNA synthetases were investigated with the use of human and mouse normal and leukemic leukocyte cell lines. Differential centrifugation of homogenized cell suspensions produced three pelleted subcellular fractions (nuclear and membrane, microsomal, and postribosomal) and a supernatant fraction. Each fraction was assayed for both methyltransferase activity and synthetase activity. The largest amounts, 40-50%, of total methyltransferase and synthetase activities were localized in either the microsomal or the postribosomal fractions, depending on cell type. In addition, the highest specific activities of these two enzyme systems were found to be present in the microsomal and postribosomal fractions. The psotribosomal fraction from leukemic leukocytes had a methyltransferase specific activity higher than that of the microsomal fraction, while the same two fractions of normal leukocytes had approximately equal activities. Specific activities of aminoacyl-tRNA synthetases were found to be approximately equal for these two fractions, whether they were from normal or leukemic leukocytes. The activity of tRNA methyltransferases and synthetases within the postribosomal fraction of the cytoplasm suggests the existence of high-molecular-weight enzyme complexes for the modification as well as the aminoacylation of tRNA.

Amino Acyl-tRNA Synthetases↗

PubMed1976

Interaction of aminoacyl-tRNA synthetases and tRNA: positive and negative cooperativity of their active centres.

The influence of tRNA on the kinetics of PP-ATP exchange and aminoacyl-tRNA formation catalysed by leucyl-, phenylalanyl-, and tryptophanyl-tRNA synthetases has been investigated. These enzymes were chosen because they belong to three main classes of quaternary structure alpha1, alpha2beta2 and alpha2, respectively. The present paper shows that the investigated synthetases manifest kinetic cooperativity of the active centres which is negative in the case of AAA formation and positive in the case of leucyl- and tryptophanyl-tRNA synthesis. The obtained data were interpreted with the aid of the trigger model of the enzyme.

Adenosine Triphosphate↗

PubMed2024

Self-quenched tRNA reporters for imaging tRNA-derived RNA biogenesis.

tRNA-derived small RNAs (tDRs) are an emerging class of small non-coding RNAs that play crucial roles in various cellular processes. However, there is a paucity of data on their sub-cellular localization due to a lack of tools and reagents to image tDRs. Imaging tDRs remains challenging due to the similar sequences between tDR and its parent tRNA. Here, we describe an innovative tool for studying the formation and localization of tDRs in various biological processes using a self-quenched tDR biogenesis reporter. This method utilizes a full-length tRNA molecule conjugated with both fluorescence and quencher groups at 5'- and 3'- ends. In its intact state, the fluorescence is quenched. Upon cleavage by specific ribonucleases and strand separation, the fluorescence becomes detectable, allowing real-time imaging of tDR biogenesis. This protocol details the design, synthesis, and application of this reporter, including transfection procedures and imaging techniques. The method offers a powerful approach for investigating tDR dynamics in living cells, providing insights into their roles in cellular processes and stress responses.

RNA, Transfer↗

PubMed1975

Functional differences in protein synthesis between rat liver tRNA and tRNA from Novikoff hepatoma.

Synthesis of ovalbumin in fragmented oviduct magnum explants of immature, estrogen-stimulated chicks has been studied in the presence of exogenous tRNA. tRAN from Novikoff hepatoma specifically inhibited ovalbumin synthesis, determined by precipitation with antisera. In addition, the major protein(s) synthesized in the presence of hepatoma tRNA had higher electrophoretic mobility than ovalbumin, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. tRNAs from rat liver, rooster liver, and hen oviduct did not affect ovalbumin synthesis, although oviduct tRNA is stimulatory during the earlier stages of estrogen stimulation.

Animals↗

PubMed1977

Aminoacylation of tRNA Trp from beef liver, yeast and E. coli by beef pancrease tryptophan-tRNA ligase. Stoichiometry of tRNATrp binding.

The Michaelis constants and the maximum velocities in the aminoacylation reaction of tRNATrp from beef liver, yeast and E. coli by pure beef pancreas tryptophan-tRNA ligase show that this mammalian enzyme recognizes and charges the two eucaryotic tRNAs with the same efficiency. The rate of aminoacylation of the procaryotic tRNATrp by the enzyme is three orders of magnitude lower. The pH optimum of aminoacylation is 8 for both eucaryotic tRNAs. The optimum magnesium concentration is different. The rate is maximum when magnesium concentration is stoichiometric to ATP concentration for tRNATrp from beef liver and 10 mM above ATP concentration for tRNATrp from yeast. The number of binding sites on the enzyme for the two eucaryotic tRNAs has been measured by equilibrium filtration on Sephadex G-100 and found equal to two.

Adenosine Triphosphate↗

PubMed1975