Flipping of the Ribosomal A-Site Adenines Provides a Basis for tRNA Selection

• Computational modeling and simulation combined with NMR and fluorescence experiments inform and resolve a controversy on tRNA selectivity in the ribosome.
• Free energy calculations of adenine flipping for A-site adenines 1492/1493 indicate that 3–4 kcal/mol selectivity arises from coupling adenines with the mRNA–tRNA minihelix.
• The strain associated with moving from the more open form and the closed form accounts for nearly 4 kcal/mol in selectivity.
• The free energy cost of keto to enol tautomerization is about 7 kcal/mol in the context of the ribosome with bound tRNA and mRNA, thus suggesting that it is unlikely.

Ribosomes control the missense error rate of ~ 10− 4 during translation though quantitative contributions of individual mechanistic steps of the conformational changes yet to be fully determined. Biochemical and biophysical studies led to a qualitative tRNA selection model in which ribosomal A-site residues A1492 and A1493 (A1492/3) flip out in response to cognate tRNA binding, promoting the subsequent reactions, but not in the case of near-cognate or non-cognate tRNA. However, this model was recently questioned by X-ray structures revealing conformations of extrahelical A1492/3 and domain closure of the decoding center in both cognate and near-cognate tRNA bound ribosome complexes, suggesting that the non-specific flipping of A1492/3 has no active role in tRNA selection. We explore this question by carrying out molecular dynamics simulations, aided with fluorescence and NMR experiments, to probe the free energy cost of extrahelical flipping of 1492/3 and the strain energy associated with domain conformational change. Our rigorous calculations demonstrate that the A1492/3 flipping is indeed a specific response to the binding of cognate tRNA, contributing 3 kcal/mol to the specificity of tRNA selection. Furthermore, the different A-minor interactions in cognate and near-cognate complexes propagate into the conformational strain and contribute another 4 kcal/mol in domain closure. The recent structure of ribosome with features of extrahelical A1492/3 and closed domain in near-cognate complex is reconciled by possible tautomerization of the wobble base pair in mRNA–tRNA. These results quantitatively rationalize other independent experimental observations and explain the ribosomal discrimination mechanism of selecting cognate versus near-cognate tRNA.

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