Michel Renaud

The yeast aminoacyl-tRNA synthetases. Methodology for their complete or partial purification and comparison of their relative activities under various extraction conditions.

Several fractionation steps are described which can be applied to the partial purification of the 20 aminoacyl-tRNA synthetases from commercial bakers yeast. Comparative experiments performed in the presence or absence of protease inhibitors revealed that some enzymes prepared in the presence of the inhibitor exhibit much higher specific activities than the proteins extracted in the absence of the inhibitor. The methodology reported can be used for the simultaneous preparation of several pure aminoacyl-tRNA synthetases. As examples, the large scale purification of phenylalanyl-and valyl-tRNA synthetases are described.

Quantitative study of the ionic interactions between yeast tRNAVal and tRNAPhe and their cognate aminoacyl-tRNA ligases

A general characteristic of the interactions between the tRNAs and the aminoacyl-tRNA ligases is that they are lowered by ionic strength. One explanation is that ionic strength weakens ionic interactions existing between phosphate groups of the tRNA and positive charges of the enzyme. A quantitative study based on this assumption was undertaken by Loftfield [l] . Using the Debye-Hiickel law, he found 2 to 4 charges interacting with the same number of positive ones. Since the Debye-Htickel law is valid only for small ions, the validity of these conclusions is questionable. A second explanation for the salt effect was proposed by Yarus [2] who postulated that the interactions between tRNA and enzyme required a conformational change and that this change was inhibited by salt. We report here a study of the affinity of tRNAVa’ and tRNAPhe for their cognate aminoacyl-tRNA ligases as a function of ionic strength under conditions where conformational changes can be considered as negligeable. Our results agree with the model of interaction between nucleic acids and protein developed by Daune [3-51. Our data are consistent with the existence of 5-6 phosphate groups interacting with the same number of positive charges on the enzyme. The participation of ionic interactions in the binding of the tRNA by its cognate aminoacyl-tRNA ligase would be about 40 to 60% of the total energy of interaction under usual conditions. This model is also sufficient to explain the increase in tRNA affinity for its aminoacyl-tRNA ligase in the presence of methanol

Cross-blot and cross-dot system: A high-performance system for the detection of antigen-antibody complexes on nitrocellulose

We present a reliable, simple, and quick system for screening antibody-antigen complexes on nitrocellulose. The apparatus necessary for this system is inexpensive and easy to use, and it can be adapted to blot or dot analysis without any modification. The number of antibody-antigen combinations that can be tested in one experiment ranges from 25 to 31 for blot analysis and from 345 to 600 for dot analysis. This system also offers numerous experimental advantages: it makes it possible to estimate with only one experiment the contribution of the different reaction stages to background noise and so allows unambiguous interpretation of the antibody-antigen reaction. Furthermore, this system can be used for any hybridization experiment on nitrocellulose.

Study of the interaction between yeast tRNAPhe and yeast phenylalanyl-tRNA synthetase by monochromatic ultraviolet irradiation at various wavelengths Advantages and limits of the method

The interactions between yeast tRNAphe and phenylalanyl-tRNA synthetase were studied by analysis of the covalent adducts obtained upon monochromatic ultraviolet irradiation at different wavelengths (248, 282, 292, 302 and 313 nm). The high extent of inactivation of phenylalanyl-tRNA synthetase, together with the partial modification of tRNA, as well as the peculiar instability of most of the covalent bonds formed upon irradiation constitute severe limitations to the use of the technique and to the interpretation of the results. These disadvantages led us to select an irradiation wavelength of 248 nm and to use only mild isolation procedures allowing a good recovery of the covalent adducts formed. Seven major tryptic peptides of the enzyme were found to be cross-linked to tRNAPhe whereas six major T1-oligonucleotides were covalently linked to the protein, among these, the three cross-linked oligonucleotides previously described by Shoemaker and Schimmel (J. Biol. Chem. 250 (1975) 4440-4444) in the same system. The difference in the number of covalently linked oligonucleotides is discussed in the light of the instability of the covalent linkages. The localization of the six oligonucleotides at the inside of the two branches forming the L-shaped tRNA molecule is similar to that observed in the yeast valine system (Renaud et al., Eur. J. Biochem. 101 (1979) 475-483) and is consistent with the interaction model previously described (Rich and Schimmel, Nucl. Acids Res. 4 (1977) 1649-1665 and Ebel et al. in Transfer RNA: structure, properties and recognition, (1979) pp. 325-343 Cold Spring Harbor Laboratory, NY). The occurrence of covalent cross-linking upon irradiation in the tryptophan absorption band (302 nm) strongly suggests the participation of this residue in the stabilization of the tRNA enzyme complex.

Interaction between tRNA and Aminoacyl-tRNA Synthetase in the Valine and Phenylalanine Systems from Yeast

Much work has been devoted to studies of the interaction between tRNAs and aminoacyl-tRNA synthetases. The aim was to understand the mechanisms that govern the specificity of the tRNA aminoacylation reaction (for recent general reviews, see Kisselev and Favorova 1974; Soll and Schimmel 1974; Goddard 1977; Ofengand 1977). In the early days, indirect approaches to this problem were used; recently, direct approaches have been introduced. The indirect approaches included perturbing the primary structure of tRNA by chemical or genetic means, excising small sequences in tRNA and correlating these perturbations with the activity of the modified molecules, or comparing the sequences of different tRNAs recognized by the same aminoacyl-tRNA synthetase. The direct approaches include measuring the inhibition of the tRNA aminoacyl-tRNA synthetase interaction caused by various factors (e.g., oligonucleotides or ionic strength) or studying the shielding of some areas of the tRNA by the aminoacyl-tRNA synthetase against RNases, oligonucleotides, or chemicals; they also include measurements by physical means of the thermodynamic parameters involved in the interaction between the aminoacyl-tRNA synthetase and intact or fragmented tRNA and the determination of the contact zones between the two macromolecules by cross-linking methods (for additional details, see reviews by Loftfield 1972; Schimmel 1977; Smith 1977). The interpretation of the results of most of these approaches has been greatly facilitated by knowledge of the structures of tRNA (Rich and RajBhandary 1976; Dirheimer et al., this volume) and aminoacyl-tRNA synthetase (Irwin et al. 1976; Zelwer et al. 1976; Winter and Hartley 1977). At this stage, however, this...