Excluded Volume of EF-Tu Greatly Reduces the Barrier to Accommodation for aa-tRNA in the Ribosome

Abstract

Functional rearrangements in biomolecular assemblies result from diffusion across an underlying energy landscape. During the process of accommodation in the ribosome, aminoacyl-tRNA (aa-tRNA) displaces the encoded amino acid ∼90 A from outside of the ribosome (A/T state) to the peptidyltransferase center (A/A state), where it is added into the nascent protein chain. This process takes place after an initial selection step consisting of GTP hydrolysis of ternary complex (aa-tRNA/EF-Tu/ GTP), which releases aa-tRNA from EF-Tu. High-resolution ribosomal crystal structures allow for atomistic models that accurately account for the configurational entropy of the aa-tRNA as it undergoes accommodation constrained by the excluded volume of both the accommodation corridor of the ribosome and EF-Tu. These structural models can be combined with approximate energy landscapes (structure-based models SBM) that encode the accommodated state as the global energy minimum. We have performed extensive characterization of the interplay between configurational entropy and effective enthalpy during the accommodation process through umbrella sampling of the aa-tRNA elbow displacement. These simulations highlight a structural role for EF-Tu in accommodation beyond initial selection. The excluded voume of EF-Tu reduces the stability of the A/T basin, lowering the barrier to aa-tRNA elbow accommodation by as much as 10kBT, depending on parameterization, and shifts the equilibrium towards to the A/A basin. This result, along with results showing that near-cognate aa-tRNA raises the accommodation barrier relative to cognate, suggests the possibility that the kinetic proofreading step is closely tied to EF-Tu dissociation.