Abstract
It has long been recognized that the thermodynamics of mRNA–tRNA base pairing is insufficient to explain the high fidelity and efficiency of aminoacyl-tRNA (aa-tRNA) selection by the ribosome. To rationalize this apparent inconsistency, Hopfield proposed that the ribosome may improve accuracy by utilizing a multi-step kinetic proofreading mechanism. While biochemical, structural and single-molecule studies have provided a detailed characterization of aa-tRNA selection, there is a limited understanding of how the physical–chemical properties of the ribosome enable proofreading. To this end, we probe the role of EF-Tu during aa-tRNA accommodation (the proofreading step) through the use of energy landscape principles, molecular dynamics simulations and kinetic models. We find that the steric composition of EF-Tu can reduce the free-energy barrier associated with the first step of accommodation: elbow accommodation. We interpret this effect within an extended kinetic model of accommodation and show how EF-Tu can contribute to efficient and accurate proofreading.
Highlights
It has long been recognized that the thermodynamics of messenger RNA (mRNA)–tRNA base pairing is insufficient to explain the high fidelity and efficiency of aminoacyl-tRNA selection by the ribosome
GTP hydrolysis is followed by Pi release, which signals a conformational change in EF-Tu and aminoacyl-transfer RNA (aa-tRNA) accommodation
Explicitsolvent models provide a detailed description of RNA and protein energetics, which has enabled the simulation of folding of tetraloops[41,42] and the calculation of free-energies for isolated regions of the ribosome[43,44], as well as the evaluation of enthalpies[45] and diffusion coefficients[46] for fully-assembled ribosomes
Summary
It has long been recognized that the thermodynamics of mRNA–tRNA base pairing is insufficient to explain the high fidelity and efficiency of aminoacyl-tRNA (aa-tRNA) selection by the ribosome. Structural and single-molecule studies have provided a detailed characterization of aa-tRNA selection, there is a limited understanding of how the physical–chemical properties of the ribosome enable proofreading To this end, we probe the role of EF-Tu during aa-tRNA accommodation (the proofreading step) through the use of energy landscape principles, molecular dynamics simulations and kinetic models. Cryoelectron microscopy (cryo-EM) and X-ray crystallography have elucidated the conformational properties of these biochemically identified states and have provided detailed insights into steps preceding and following accommodation[20,21,22,23,24,25,26] To complement these static snapshots, single-molecule FRET (smFRET) studies have shed light on the dynamics of individual aa-tRNA molecules during initial selection[27] and accommodation[28]. The A/T-configured aa-tRNA is shown in yellow
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