The Ribosomal E Site at Low Mg2+: Coordinate Inactivation of Ribosomal Functions at Mg2+Concentrations Below 10 mM and its Prevention by Polyamines

Abstract

Under standard conditions (Mg²⁺/150 mM NH₄ ⁺) ribosomes can quantitatively participate in tRNA binding at Mg²⁺ concentrations of 12 to 15 mM. The overall poly(U)-directed Phe incorporation and the extent of tRNA binding to either P,E or A sites decrease in a parallel manner when the Mg²⁺ concentration is lowered below 10 mM. At 4 mM the inactivation amounts to about 80%. The coordinate inactivation of all three binding sites is accompanied by an increasing impairment of the ability to translocate A-site bound AcPhe-tRNA to the P site. The translocation efficiency is already reduced at 10 mM Mg²⁺, and is completely blocked at 6—8 mM. The severe inactivation seen at 6 mM Mg²⁺ vanishes when the polyamines spermine (0.6 mM) and spermidine (0.4 mM) are present in the assay; tRNA binding again becomes quantitative, the total Phe synthesis even exceeds that observed in the absence of polyamines by a factor of 4. In the presence of polyamines and low Mg²⁺ (3 and 6 mM) two essential features of the allosteric three-site model (Rheinberger and Nierhaus,J.Biol. Chem. 261, 9133 (1986)) are demonstrated. 1) Deacylated tRNA is not released from the P site, but moves to the E site during the course of translocation. 2) Occupation of the E site reduces the A site affinity and vice versa (allosteric interactions between E and A sites). The quality of an in vitro system for protein synthesis can be assessed by two criteria. First, the incubation conditions must allow a near quantitative tRNA binding. Secondly, protein synthesis should proceed with near in vivo rate and accuracy. The 3 mM Mg²⁺/NH₄ ⁺/polyamine- system seems to be the best compromise at present between these two requirements.