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Alkylation in beef tRNATrp of phosphodiester bonds by ethylnitrosourea and of N-7 in guanosines and N-3 in cytidines by dimethyl sulfate and carbethoxylation of N-7 in adenosines by diethyl pyrocarbonate were investigated under various conditions. This enabled us to probe the accessibility of tRNA functional groups and to investigate the structure of tRNAT'p in solution as well as its interactions with tryptophanyl-tRNA synthetase.The phosphate reactivity towards ethylnitrosoureaof unfolded tRNA was compared to that of native tRNA. The pattern of phosphate alkylation of tRNATrp is very similar to that found with other tRNAs studied before using the same approach with protected phosphates mainly located in the D and TY arms. Base modification experiments showed a striking similarity in the reactivity of conserved bases known to be involved in secondary and tertiary interactions. Differences are found with yeast tRNAPh" since beef tRNATrp showed a more stable D stem and a less stable T Y stem.When alkylation by ethylnitrosourea was studied with the tRNATrp. tryptophanyl-tRNA synthetase complex we found that phosphates located at the 5' side of the anticodon stem and in the anticodon loop were strongly protected against the reagent. The alkylation at the N-3 position of the two cytidines in the CCA anticodon was clearly diminished in the synthetase . tRNA complex as compared with the modification in free tRNATrp; in contrast the two cytidines of the terminal CCA in the acceptor stem are not protected by the synthetase. The involvement of the anticodon region of tRNATrp in the recognition process with tryptophanyl-tRNA synthetase was confirmed in nuclease S1 mapping experiments.After a quarter of a century of intensive work, the role of tRNA in protein biosynthesis has been well established [l]. However this is not the only cellular role played by these molecules. Transfer RNAs are also involved in processes as different as the biosynthesis of the bacterial cell wall, the regulation of transcription through the mechanism of attenuation and the expression of retroviruses [2, 31.The primary structure of several hundred tRNAs is known [4], while the tridimensional structure of only two elongator tRNAs is known at high resolution [5-91. These structures have been determined by X-ray diffraction of tRNA crystals. However, only some tRNAs are able to give crystals suitable for X-ray diffraction studies, so that other techniques for studying tRNA structure in solution have been developed. In general good agreement is obtained between results of diffraction and solution studies [lo-121. One of these techniques for the study of tRNA in solution consists in alkylating unspecifically the phosphate groups of the tRNA backbone with the reagent N-ethylnitrosourea. This substance will modify only those phosphate groups not shielded by the three-dimensional structure of the molecule [lo]. Other reagents used in recent years for the modification of nucleic acids are dimethyl sulfate which alkylates the N-3 position of cytidines and ...

The aminoacylation reaction catalyzed by the dimeric tryptophanyl-tRNA synthetase from beef pancreas was studied under pre-steady-state conditions by the quenched-flow method. The transfer of tryptophan to tRNATrp was monitored by using preformed enzyme-bis(tryptophanyl adenylate) complex. Combinations of either unlabeled or L-[14C]tryptophan-labeled tryptophanyl adenylate and of aminoacylation incubation mixtures containing either unlabeled tryptophan or L-[14C]tryptophan were used. We measured either the formation of a single labeled aminoacyl-tRNATrp per enzyme subunit or the turnover of labeled aminoacyl-tRNATrp synthesis. Four models were proposed to analyze the experimental data: (A) two independent and nonequivalent subunits; (B) a single active subunit (subunits presenting absolute "half-of-the-sites reactivity"); (C) alternate functioning of the subunits (flip-flop mechanism); (D) random functioning of the subunits with half-of-the-sites reactivity. The equations corresponding to the formation of labeled tryptophanyl-tRNATrp under each labeling condition were derived for each model. By use of least-squares criteria, the experimental curves were fitted with the four models, and it was possible to disregard models B and C as likely mechanisms. Complementary experiments, in which there was no significant excess of ATP-Mg over the enzyme-adenylate complex, emphasized an activator effect of free L-tryptophan on the rate of aminoacylation. This result disfavored model A. Model D was in agreement with all data. The analyses showed that the transfer step was not the major limiting reaction in the overall aminoacylation process.

The reaction rates between dansyl chloride and water, amino acids or peptides are studied as a function of pH and temperature. The rate of hydrolysis of dansyl chloride is constant and low up to pH 9.5 and above this pH it increases rapidly. The various reactive groups of amino acids and peptides react with dansyl chloride in their unprotonated form. It is shown that a compromise for optima conditions of dansylation (pH and temperature) may be found, taking into account the rate of hydrolysis and the pK of the group to be dansylated. A complete chromatographic separation on Silica gel G thin layer plates of dansyl amino acids is proposed using 4 solvents. The quantitative recovery of the separated derivatives is described. The study of the acid hydrolysis of dansylated proteins shows that the release of the dansyl derivatives from the peptide chain is faster than that of free amino acids; it also shows that the destruction of dansyl amino acids occurs rather rapidly. Therefore a hydrolysis time of 4 hours (110°) is suggested, except in the special cases of the release of dansyl valine, dansyl leucine or dansyl isoleucine which needs 18 hours of hydrolysis. For quantitative estimation of each dansyl derivative a correction factor is given, taking into account the loss during hydrolysis, the recovery from the plates and the individual fluorescence of any dansyl derivative as compared to the fluorescence of a reference compound. The general conditions described in this work require 1 nmole of material for qualitative determination of the N‐terminal amino acids, and 5–10 nmoles for their quantitative estimation. Some examples (α‐chymotrypsin, trypsinogen, ribonuclease, lysozyme, aspartokinase and triose phosphate dehydrogenase) illustrate the efficiency of the method.