Mechanical forces acting on the ribosome can alter the speed of protein synthesis, indicating that mechanochemistry can contribute to translation control of gene expression. The naturally occurring sources of these mechanical forces, the mechanism by which they are transmitted 10 nm to the ribosome's catalytic core, and how they influence peptide bond formation rates are largely unknown. Here, we identify a new source of mechanical force acting on the ribosome by using in situ experimental measurements of changes in nascent-chain extension in the exit tunnel in conjunction with all-atom and coarse-grained computer simulations. We demonstrate that when the number of residues composing a nascent chain increases, its unstructured segments outside the ribosome exit tunnel generate piconewtons of force that are fully transmitted to the ribosome's P-site. The route of force transmission is shown to be through the nascent polypetide's backbone, not through the wall of the ribosome's exit tunnel. Utilizing quantum mechanical calculations we find that a consequence of such a pulling force is to decrease the transition state free energy barrier to peptide bond formation, indicating that the elongation of a nascent chain can accelerate translation. Since nascent protein segments can start out as largely unfolded structural ensembles, these results suggest a pulling force is present during protein synthesis that can modulate translation speed. The mechanism of force transmission we have identified and its consequences for peptide bond formation should be relevant regardless of the source of the pulling force.
mutant derivatives are investigated with optical tweezers, steered molecular dynamics (SMD) simulations and single-molecule FRET (smFRET). With smFRET, we show that base triples at one end (formed by loop 1 and stem 2) of hTR-PK promote the formation of those at the distal end (formed by stem 1 and loop 2), and thereby limit the flexibility of the distal loop (loop 2). The coordination between base triples is therefore responsible for the single-step unfolding event of the structure. By contrast, smFRET experiments identify a compact intermediate structure before hTR-PK is completely disrupted by the ribosome. SMD simulations further reveal that as the hTR-PK loop 2 attaches to the positively charged residues of ribosomal protein S3, base triples facilitate formation of the unfolding intermediate of hTR-PK. When the base triples are disrupted by mutations (the delta-triple mutant), the compact unfolding intermediate can no longer be seen in SMD simulations and smFRET experiments (that is, the mutant exhibits single-step unfolding). The delta-triple mutation also results in a dramatic drop in FS efficiency from 50% to 0%. Our study demonstrates the importance of a base triple-stabilized unfolding intermediate in PK-induced FS.
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