Distinct Steps in the Specific Binding of tRNA to Aminoacyl-tRNA Synthetase. Temperature-Jump Studies on the Serine-Specific System from Yeast and the Tyrosine-Specific System from Escherichia coli

Abstract

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 ...

Detection of Noncovalent tRNA·Aminoacyl-tRNA Synthetase Complexes by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

“…Therefore, the MS experiments suggest that yeast TyrRS besides the most preferred complex ␣ 2 ⅐tRNA may also form symmetrical complexes which cannot be detected by gel retardation assay due to their lower stability. The same stoichiometry of the complexes detected for the serine system is consistent with biochemical experiments indicating the binding of two molecules of tRNA to the dimeric enzyme with different affinities (53).…”

High Affinity Binding of the Receptor-associated Protein D1D2 Domains with the Low Density Lipoprotein Receptor-related Protein (LRP1) Involves Bivalent Complex Formation

“…The overall equilibrium binding constant for Scheme 1(K A1 ) is calculated as described Equation 1 (51,52),…”

Yeast seryl tRNA synthetase: interactions between the ATP binding site and the sites for tRNASerand L-serine

“…It is calculated from data on the binding of tRNASer to seryl tRNA synthetase (7,17,18) that under the conditions of the nuclease protection studies about 90 % of the tRNA were bound to seryl tRNA synthetase as first, and less than 5 % as second tRNA. Because of the high synthetase concentration only a small amount of tRNA was free.…”

“…Accordingly it is the relationship between the binding of this first tRNASer and of the cosubstrates which was investigated. On the basis of the fluorescence relaxation kinetics it is assumed that this first tRNASer is distributed to one third in a primary complex with the synthetase, ES, and to two thirds in a rearranged complex, ES+ (18). Accordingly, tRNASer is protected in both, the ES and the ES+ complexes.…”

“…Apparently, there exist not only different states of the synthetase-tRNASer complex (7,18) but also several distinct states of the synthetase when the small ligands ATP, serine and their-reaction products are bound to the enzyme. Further biochemical and physicochemical work will have to define such states more closely.…”