We have used site-directed mutagenesis of the EcoRV restriction endonuclease to change amino acid side chains that have been shown crystallographically to be in close proximity to the scissile phosphodiester bond of the DNA substrate. DNA cleavage assays of the resulting mutant proteins indicate that the largest effects on nucleolytic activity result from substitution of Asp74, Asp90, and Lys92. We suggest on the basis of structural information, mutagenesis data, and analogies with other nucleases that Asp74 and Asp90 might be involved in Mg2+ binding and/or catalysis and that Lys92 probably stabilizes the pentacovalent phosphorus in the transition state. These amino acids are part of a sequence motif, Pro-Asp...Asp/Glu-X-Lys, which is also present in EcoRI. In both enzymes, it is located in a structurally similar context near the scissile phosphodiester bond. A preliminary mutational analysis with EcoRI indicates that this sequence motif is of similar functional importance for EcoRI and EcoRV. On the basis of these results, a proposal is made for the mechanism of DNA cleavage by EcoRV and EcoRI.

The equilibria between the elongation factor Tu . GTP complex (EF-Tu . GTP) from Escherichia coli and tyrosyl-tRNATY' from E. coli as well as phenylalanyl-tRNAPhe and seryl-tRNASer from yeast were studied using a novel procedure, which takes advantage of the protective effect of ternary complex formation on the stability of the aminoacyl bond against non-enzymatic hydrolysis. At 25 "C and at pH 7.4 tyrosyl-tRNATY', phenylalanyl-tRNAPhe and seryl-tRNASe' are bound with binding constants of 0.7 x lo7 M-', 5.0 x lo7 M-' and 0.5 x lo7 M-' respectively. The binding of aminoacyl-tRNA to EF-Tu . GTP has a negative d H of the order of 10 kcal/mol(42 kJ/mol). Complex formation is dependent on ionic strength: with 0.1 M KC1 K,,, = 0.5 x lo7 M-' , w' ith 0.5M KCl K,,, = 0.2 x lo7 M-' was determined for the binding of Tyr-tRNATY'.

The kinetics of the interaction of tRNASer and seryl-tRNA synthetase from yeast as well as of tRNATy' and tyrosyl-tRNA synthetase from Eschevichia coli have been investigated by temperaturejump 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 (k:2, kal), which is transformed in a fast reaction to the final 1 : 1 complex (ki3, ki2). At pH 7.2 with 0.1 M KCI the rate constants are: k:2 = 2 . 7~ lo8 M-' s-'; ki1 = 220 s -l ; ki3 = 760 s K ' ; ki2 = 330 s-'. The 1 : 1 complex can bind a second tRNA. At pH 7.2 without added salt the rate constants are: k:: = 0 . 9~ lo8 M -' s K ' ; k:: = 270 s-'; k:: = 120 sK1; k& = 1250 sK1.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: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.The specificity of the interaction between tRNA and synthetase is one of the essential prerequisites for translational fidelity. Since this interaction requires the mutual recognition of two macromolecules, it seems attractive to differentiate between a recombination and a recognition step. Such hypotheses have been mentioned by several authors (e.g. [l -41) and have been discussed by Eigen as early as 1964 in a quite general way [4a].Stopped-flow investigations on the kinetics of tRNA binding to aminoacyl-tRNA synthetases have shown that the recombination process is close to diffusion controlled [2,.5,6]. Furthermore, evidence was presented that the association is not followed by a slow rearrangement; a fast one, however, with a half-life shorter than that accessible to the time resolution of the stopped-flow technique, could not be excluded [2].An outline of this work was reported at the 10th FEBS Meetlng, Paris, July 1975, and at the 5th International Blophyslcs Congress, Copenhagen, August 1975 It was the purpose of the work reported in this paper to investigate the specific interaction of two tRNAs with their cognate synthetase by temperaturejump techniques in order to search for fast reactions succeeding the recombination of tRNA and synthetase. MATERIALS AND METHODS SynthetasesSeryl-tRNA synthetase was prepared from commercial baker's yeast as outlined in [7]. It had a specific activity of 350 units/mg protein under conditions of [S] and was homogeneous by dodecylsulfate gel electrophoresis. A r2/m' at 280 nm was 1.01 [7].Tyrosyl-tRNA synthetase was prepared from E. coli MRE 600 (Merck, Darmstadt) using a DEAEcellulose (DE-52, Whatman) chromatography (0.0 -0.4 M KC1 gradient in 0.03 M potassium phosphate pH 7.2,20 "/, glycerol, 0.5 mM EDTA, 0.5 mM dithioerythritol, 0.1 mM ...