Association equilibria have been determined in the ternary system uridyl triplets (T)-ribosomal protein SI (S)-ribosomes (Rb) depleted of SI at 6 and 10 mM Mg2+. For 1:1 stoichiometry of reactants, four thermodynamically independent equilibria characterize the ternary system. The binary interaction Rb + T was studied by following the fluorescence quenching of labeled ribosomes by added T. The Rb + T association constant for UpUpUp triplets was 10-20-fold greater than for ApUpG triplets. The interaction Rb + S was studied by following the changes in fluorescence anisotropy when labeled SI reacted with ribosomes. The remaining two independent equilibrium constants (for S + T and RbT + S) were obtained from fits to observed anisotropy measurements when varying amounts of T were added to a solution of ribosomes and fluorescently labeled SI. This indirect procedure allows one to measuee S + T binding, an association that is difficult to determine directly. Over the concentration interval 5-10 mM Mg2+, the association constant for Rb + S increases with the sixth power of [Mg2+], whereas the association constant for S + T decreases approximately 2-fold as Mg2+ is increased from 6 to 10 mM Mg2+. T binds to Rb more tightly at 10 mM than at 6 mM Mg2+. When SI is bound to Rb, however, at 10 mM Mg2+ the binding constant for T is decreased 10-fold and the Mg2+ dependence is reversed. These interactions can be described in terms of coupling free energies. For the ternary complex, three linearly independent coupling free energies can be written. These excess functions simply show by how much we err in estimating the overall free-energy change for formation of the RbST complex from free-energy changes for the formation of various binary fragments. One of the ternary coupling free energies, ΔG°r,st (=ΔG°rst - °G°RT - °G° Rs), is positive (anti-cooperative interaction) at 10 mM Mg2+ but negative at 6 mM Mg2+, primarily because of the sensitivity of ΔG°rs to [Mg2+], Thus, at 6 mM Mg2+, prior binding of S1 to the ribosome enhances binding of the triplet to form the ternary complex, but at 10 mM Mg2+, SI binding to Rb destabilizes subsequent binding of the triplet. More elaborate models that assumed multiple sites for T on S and Rb were used to fit the data; however, both the anisotropy data and the results for triplet quenching of ribosome fluorescence were in accord with a simple description where the binding of the triplets to the ribosome appeared to be to a single site. If there are multiple sites for triplet binding, the binding sites show little interaction or heterogeneity over the concentration ranges studied. Our results lend support to assigning primary importance to protein-protein interactions in the binding of S1 to the ribosome. Although S1 may well function as an unwinding protein, it can profoundly affect the binding of nonhelical trinucleotides to the ribosome.
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