Claudine Bollack

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 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.

Synthesis of ferritin in cultured hepatoma cells

Ferritin is the major intracellular site for iron storage [ 11. It has many useful properties and advantages, despite the fact that it is not an enzyme, for studying the regulation of protein synthesis and turnover in mammalian tissues [ 2-41. In the present report the synthesis of ferritin has been studied in a cell line of a hepatoma tissue culture called ‘HTC cells’. These cells were derived from a malignant tumor of rat liver parenchymal cells (Morris hepatoma 7288 C) and adapted to growth in cell culture by Thompson et al. [S] . The outstanding characteristic of HTC cells is their response to the adrenal steroids. The synthesis of ferritin has been studied in suspension cultures of HTC cells as well as in cell free extracts incubated in vitro by measuring the incorporation of [3 H] leucine into proteins. The labelled ferritin was isolated by antibody precipitation after a preliminary purification in the presence of carrier rat liver ferritin. Apoferritin was synthesized to the same rate by the whole cells and by the cell free extracts but only a slight increase in ferritin protein concentration following iron treatment was observed. No measurable effect of glucocorticoids was observed on ferritin synthesis. Thus, it becomes interesting to retain ferritin as an internal control protein in the study of hormonal induction of tyrosine aminotransferase (TAT) in HTC cells. 2. Material and methods

Regulation of tyrosine aminotransferase synthesis in vitro by mRNA and soluble factors from hepatoma tissue culture cells.

In the last few years, evidence has been accumulated showing that gene expression in eukaryotic cells is not only regulated at the level of transcription, but also at post-transcriptional steps (reviewed [I]). Tyrosine aminotransferase (TAT, EC 2.6.1.5.) has several distinctive features which have made this enzyme a convenient model for investigation of protein synthesis in hepatoma cells [2,3] . As an approach to the problem of regulation of TAT by glucocorticoids, we investigated the synthesis of the enzyme in cell-free systems where the translational control of specific mRNAs and the mechanisms in post-translational processing could be studied in detail. In this paper, we have examined the in vitro synthesis of TAT in a homologous cell-free system from dexamethasone-induced or uninduced hepatoma tissue culture (HTC) cells. Since the rate of TAT synthesis is greater in extracts prepared from induced cells than from uninduced cells, we tried to determine which cellular components are required for the expression of this increased rate of synthesis. Our results show that induction enhances largely the TAT synthetic activity of the polysomal fraction; in addition, the soluble fraction from induced cells contains a dialysable component which optimizes TAT specific mRNA translation. These results are consistent with the observations made in whole cells which suggest that enzyme induction results from an increase in enzyme-specific mRNA and that TAT synthesis is also regulated at the translational level [4] . 2. Materials and methods

Inhibition de la multiplication virale a l'aide d'acides ribonucleiques chimiquement modifies: I. Inhibition de la multiplication de Myxovirus parainfluenzae I (virus sendai) sur cellules de rein de veau

Abstract 1. The introduction of foreign ribonucleic acids into the cellular inheritance system carries with it, in the case of calf kidney cortex cells, an apparent inhibitory capacity with respect to the multiplication of a Myxovirus. 2. If the foreign RNA is heterologous to that of the host cell (e.g. yeast RNA), then, whether or not the RNA is chemically modified, the inhibition is substantial but incomplete. 3. If the foreign RNA is homologous with respect to the host cell, but modified by methylation, the inhibition is more pronounced or complete. 4. One of the possible mechanisms for this action is represented by a substance whose properties are comparable with those of the interferons.

Relations between steroid-cell contact, steroid-binding and induction of tyrosine aminotransferase

The induction of tyrosine aminotransferase by a variety of steroids was studied in cells from a hepatoma tissue culture (HTC). We have defined a class of steroids that induce TAT synthesis to a higher level than optimal inducers described earlier; these are called supra-inducers. When TAT induction is compared with the binding of the steroids to the cytoplasmic receptor or to their binding in the whole cell, a good correlation between binding in vivo of the hormone and its induction capacity can be established, whereas such a correlation was not systematically observed in vitro. A very short exposure of HTC cells to either dexamethasone or corticosterone is sufficient to induce TAT. When the inducer is removed from the culture medium a few minutes after its administration, the intracellular hormone concentration decreases very rapidly but TAT will be synthesized at its maximal rate. Thus the hormones behave as a starting signal for the optimal synthesis of the enzyme, and their presence in the culture medium is not necessary throughout the entire induction period.

Intrinsic forms of soluble and mitochondrial tyrosine aminotransferase from rat tissues.

Tyrosine aminotransferase activity has been found in cytosol and in mitochondria of a variety of tissues [l-4]. The enzyme from rat liver has been the subject of the greatest scrutiny, because of its abundance in the organ. The majority of the enzyme from this tissue is soluble and can be separated into 3 or 4 multiple forms by chromatography on hydroxylapatite [5] or carboxymethyl-Sephadex [6]. The mitochondrial enzyme is much less aburidant but seems to have a broader tissue distribution than the cytosol form [7,8] and a wider substrate spectrum [9]. The objective of the studies reported here was to gain further information about the structural and functional relationship of tyrosine aminotransferase from different tissues. The soluble and the mitochondrial enzymes were analyzed by carboxymethyl (CM)-Sephadex chromatography. The enzymes from rat kidney and rat brain were studied and compared to that of the liver which is glucocorticoid inducible [lo]. Tyrosine aminotransferase (TAT) translocated into the mitochondria was eluted in one major peak at a lower KC1 concentration than the cytosol forms. The mitochondrial enzyme is common to liver and other tissues and is regulated by a mechanism different from that of soluble tyrosine aminotransferase.