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The ribosomal E site at low Mg2+: coordinate inactivation of ribosomal functions at Mg2+ concentrations below 10 mM
H J Rheinberger1, K H Nierhaus
1Max-Planck-Institut für Molekulare Genetik, Abt. Wittmann, Berlin-Dahlem, West Germany.
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.
Insights
Magnesium (Mg2+) concentration critically affects ribosome function, impacting tRNA binding and protein synthesis. Polyamines restore ribosome activity at low Mg2+ levels, improving in vitro protein synthesis systems.
Area of Science:
- Molecular Biology
- Biochemistry
- Ribosome Function
Background:
- Ribosome function, including tRNA binding and protein synthesis, is sensitive to magnesium (Mg2+) ion concentration.
- Previous models, such as the allosteric three-site model, describe ribosome-tRNA interactions.
Purpose of the Study:
- To investigate the effect of varying Mg2+ concentrations on ribosome activity, specifically tRNA binding and polypeptide synthesis.
- To determine the role of polyamines in restoring ribosome function under suboptimal Mg2+ conditions.
- To validate key aspects of the allosteric three-site model using an optimized in vitro system.
Main Methods:
- Assessing poly(U)-directed phenylalanine (Phe) incorporation into ribosomes.
- Quantifying tRNA binding to ribosomal P, E, and A sites under different Mg2+ concentrations.
- Evaluating the effect of polyamines (spermine and spermidine) on ribosome activity at low Mg2+.
Main Results:
- Decreased Mg2+ concentration below 10 mM significantly reduces tRNA binding and Phe incorporation, with inactivation reaching 80% at 4 mM Mg2+.
- Low Mg2+ impairs the translocation of AcPhe-tRNA from the A site to the P site, completely blocking it at 6-8 mM.
- The addition of polyamines rescues ribosome activity at low Mg2+ (3-6 mM), leading to quantitative tRNA binding and a fourfold increase in Phe synthesis.
- Polyamines in low Mg2+ conditions demonstrate allosteric interactions between E and A sites and prevent deacylated tRNA release from the P site during translocation.
Conclusions:
- Ribosome function is highly dependent on Mg2+ concentration, affecting multiple steps in protein synthesis.
- Polyamines are crucial for maintaining ribosome activity and efficient protein synthesis at low Mg2+ concentrations.
- The 3 mM Mg2+/NH4+/polyamine system represents an optimized in vitro condition for protein synthesis, balancing tRNA binding and efficiency.