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 tRN^Ser and seryl-tRNA synthetase from yeast as well as of tRNA^Tyr and iyrosyl-tRNA syutlietase from Escherichia coil have been investigated by temperature-jump 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^I₁₂, k^I₂₁), which is transformed in a fast reaction to the final 1:1 complex (k¹₂₃, k¹₃₂). At pH 7.2 with 0.1 M KCI the rate constants are: k¹₁₂= 2.7 × 10⁸ M⁻¹ s⁻¹; k^I₂₁= 220 s⁻¹; k^I₂₃= 760 s⁻¹; k^I₃₂= 330 s⁻¹. The 1:1 complex can bind a second tRNA. At pH 7.2 without added salt the rate constants are: k¹¹₁₂= 0.9 × 10⁸ M⁻¹ s⁻¹; k¹¹₂₁= 270 s⁻¹; k¹¹₂₃= 120 s⁻¹; k¹¹₃₂= 1250 s⁻¹. 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: k¹¹₁₂= 1 × 10⁸ (2.5 × 10⁸) M⁻¹ s⁻¹; k¹¹₂₁= 470 (170) s⁻¹; k¹¹₂₃= 150 (530) s⁻¹; k¹¹₃₂= 1540 (720) s⁻¹. 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.