N. Befort

Factors determining the specificity of the tRNA aminoacylation reaction: Non-absolute specificity of tRNA-aminoacyl-tRNA synthetase recognition and particular importance of the maximal velocity

Summary It is generally believed that the specificity of tRNA aminoacylation results solely from a specific recognition between the aminoacyl-tRNA synthetase and the cognate tRNA. In fact, this specificity is not absolute: this is supported by the following observations (1) the existence of tRNA mischarging in homologous systems under usual aminoacylation conditions, (2) the existence of inhibitions produced by « non-cognatetRNA species in correct aminoacylation reactions, (3) the lack of specificity of AMP- and PPi- independent aminoacyl-tRNA synthetase catalysed deacylation of aminoacyl-tRNA species, (4) the isolation of complexes between aminoacyl-tRNA synthetases and non-cognate tRNA species. The affinities between aminoacyl-tRNA synthetases and non-cognate tRNA species, estimated by the Km measurements in mischarging reactions, have been found only diminished by 1 or 2 orders of magnitude as compared to the values found in specific systems, whereas the Vmax values for mischarging have been found diminished by 3 or 4 orders of magnitude. This suggests that tRNA aminoacylation depends more upon the maximal velocity of the reaction than upon the recognition between aminoacyl-tRNA synthetase and tRNA. Furthermore, we found that the recognition of a tRNA by an aminoacyl-tRNA synthetase does not seem to require the 3′ terminal part of the amino acid acceptor stem. As the importance of this part of the tRNA molecule during the aminoacylation process has been well established, it is possible that it is involved in determining the Vmax of the aminoacylation reaction, probably by positioning the 3′ terminal adenosine in the catalytic site of the enzyme. In conclusion, it appears that the specificity of the tRNA aminoacylation reaction proceeds through two discrimination mechanisms: the first one, measured by the Km, acts at the recognition level; the second one, which is more effective, is measured by the Vmax values. Competition phenomena have been observed between cognate and non-cognate tRNA species. They enhance the specificity of the tRNA aminoacylation, but their contribution to the specificity is low compared to that brought by Km and Vmax. Finally we found that a more rapid enzymatic deacylation of mischarged tRNA species (as compared to correctly charged ones) cannot be considered as a general mechanism for correction of misaminoacylation.

Purification et quelques proprietes de la phenylalanyl-tRNA synthetase de levure de boulangerie

Abstract Purification and some properties of phenylananyl-tRNA synthetase from bakers yeast Phenylalanyl-tRNA synthetase has been isolated from bakers yeast with a 600-fold purification. The different steps of the preparation are: (NH 4 ) 2 SO 4 precipitation of the 78 000 × g crude extract (between 50 and 65 % saturation), chromatography on DEAE-cellulose, CM-Sephadex C-50 and hydroxylapatite. The enzyme appears to be homogeneous on hydroxylapatite chromatography, sucrose gradient centrifugation and polyacrylamide gel electrophoresis. Molecular weight determinations by sucrose gradient centrifugation or equilibrium sedimentation studies give an average value of 220 000. Amino acid composition has been determined. No end group can be detected by the dansyl method. In the presence of either ATP and phenylalanine or tRNA Phe , the number of free thiol groups titrated with DTNB decreases. The enzyme is dissociated by 8 M urea or 1 % sodium dodecyl sulphate into two different equimolar components. The molecular weights of these 2 components were estimated to be 56 000 and 63 000, respectively, by polyacrylamide gel electrophoresis. The results suggest that the enzyme has a 4-subunits structure A 2 B 2 . The kinetics of the PP i -ATP exchange and aminoacylation reactions of tRNA Phe have been determined.

Complete complementary DNA of rat tyrosine aminotransferase messenger RNA. Deduction of the primary structure of the enzyme.

The primary structure of rat tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase; EC 2.6.1.5), a liver-specific enzyme involved in gluconeogenesis, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 2362 nucleotides long (excluding the poly(A) tail) and codes for a polypeptide of 454 amino acids with a molecular weight of 50634. Unambiguous identification was obtained by comparison of this sequence with the amino acid sequences of several peptides obtained from the purified enzyme.

Purification and characterization of rat liver tyrosine aminotransferase

Several studies have been published describing the purification and properties of L-tyrosine-2-oxoglutarate aminotransferase (EC 2.6.1 S.). The purification was first attempted from the livers of L-tyrosine treated rats [ 1,2] . Later, a more extensive purification from the livers of rats previously treated with glucocorticoid hormones was reported 13-51. In this paper, we describe a purification method for the soluble tyrosine aminotransferase (TAT) from livers of rats treated with the synthetic glucocorticoid: dexamethasone. The enzyme was purified by fractionation techniques utilizing heat treatment, chromatography on DEAE-cellulose and hydroxyapatite columns, gel-filtration on Sephadex G-200 and sedimentation in a sucrose gradient. During the whole purification procedure, the enzyme remained soluble since steps like precipitation by ammonium sulfate or concentration by freeze-drying were avoided. The overall purification factor was in the range of 30004000 with a recovery of 30-40% of the activity originally present in the crude extract. Several properties of the enzyme were studied.

Etude du complexe entre tRNAPhe et phenylalanyl-tRNA synthetase de levure

Abstract Study of the complex between tRNAPhe and phenylananyl-tRNA synthetase from yeast A stable and specific complex between the tRNAPhe and the phenylalanyl-tRNA synthetase from yeast has been isolated by sucrose gradient centrifugation. One molecule of tRNAPhe is combined with the tetrameric molecule of the enzyme. In the presence of tRNAPhe which is acylated by [14C]phenylalanine, a hydrolysis of the ester bond takes place during the isolation of the complex. This hydrolysis is strongly diminished in the absence of Mg2+. The interaction between tRNAPhe and the enzyme is strongly dependent upon the pH (optimum between 5.5 and 6.0) but is not influenced by either the increase in temperature (up to 37°) or the Mg2+. In the presence of the enzyme, the tRNAPhe is strongly protected against ribonuclease T1 action.

Dinucleoside tetraphosphate variations in cultured tumor cells during their cell cycle and growth

Asynchronous and synchronized cultures of A549 and HTC cells were used to detect possible, cell cycle or cell density specific variations in the intracellular pools of dinucleoside tetraphosphates (Ap4X). No important variations of the nucleotide pools were observed during cell growth. When HTC cells were released from mitotic arrest, a decrease by a factor of N3 Ap4X and ATP levels was observed when the cells entered the G1 phase. This decrease is essentially due to cell doubling. When A549 cells were released from an arrest at the G1/S boundary, the nucleotide pool size increased slightly during the G2 phase just before mitosis. This result is in agreement with both earlier data from our laboratory and the observed decrease in Ap4X pool after release from mitotic-arrested HTC cells. These results suggest that the Ap4X and ATP pools are only subjected to very small variations during the cell cycle, essentially in the G2 phase and after mitosis.

Effects of thyroidectomy on glucocorticoid receptors in rat liver.

Glucocorticoid and thyroid hormones regulate physiological and developmental processes in a number of tissues. Both hormones have been shown to regulate RNA and protein synthesis. They induce the synthesis of 02u globin in liver parenchymental cells [ 1,2] and of growth hormone in pituitary cells [3-61. In both cases, the presence of thyroid hormone is required for the effect of glucocorticoids. The specific effects of steroid hormones have been shown to result from the cytosol-nuclear translocation of a ho~one-receptor complex. Moreover, it has been shown [7] that the con~entra~on of estrogen receptor in uterus cytosoi is modulated by thyroid hormone. This led us to study glucocorticoid receptors in rat liver in relation with the thyroid status of the animals. Here we describe a decrease in the concentration of glucocorticoid receptors and a modification of the receptor properties after thyroidectomy.

Étude comparative des acides ribonucléiques de transfert extraits du foie de rat et de l'hépatome de Zajdela: I. — Étude du pouvoir accepteur des tRNA

Summary We have studied possible changes in tRNA level during the differentiation of normal hepatic tissue into an ascitic hepatoma, by comparing the specific activities of rat liver and Zajdela hepatoma tRNAs. The standard aminoacylation conditions found for one aminoacid could not be used for the others in charging the tRNAs obtained from the two sources, since it was found that an excess of enzyme in the reaction mixture or an extended incubation time led to a decrease of the acylation level for several tRNAs. Therefore for each aminoacid a study was performed to determine the optimal conditions for aminocylation. For some aminoacids (val, arg, pro) differences were observed in the acylation levels between liver tRNA and hepatoma tRNA, when the same enzymic extrat was used. Differences in the aminoacylation levels (for instance for tRNAgly, tRNAtyr,...) were also revealed when both enzymic preparations (from liver and hepatoma) were used on the same tRNA from liver or hepatoma.

Impairment of enzyme induction by glucocorticoids in Zajdela hepatoma cells.

Whereas glucocorticoids induce TAT, TRP, GPT in liver and only TAT in HTC cells, no hormonal effect on the synthesis of these enzymes was found in Zajdela hepatoma cells grown in vivo as an ascitic tumor, or in vitro as layer cultures. Although these cells remain uninducible, the hormone penetrates normally, but a strong decrease of the specific binding of cytosol and nuclear proteins with the hormone was observed. The impairment at the level of the hormone receptors could account for the non-inducibility of enzyme synthesis in ZHC cells.

Inhibition of viral multiplication by homologous methylated ribonucleic acids IV. Subcellular localisation after uptake into fibroblasts and relation between antiviral activity and chain length

Summary The addition of chemically modified homologous ribonucleic acids to the culture induces an inhibition of viral infection. All RNAs, modified or not, enter the cells and are preferentially located at the microsomal level. The differences observed in the antiviral properties cannot be explained by the differences in the way of the uptake or by the differences in the specific resistance of the RNAs to nucleases. On the other hand, the antiviral activity is not a peculiar property of a special class of intact methylated RNA but a general property of chemically modified polynucleotidic chains of a minimal chain length of 40 nucleotides.