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Spontaneous formation of the unlocked state of the ribosome is a multistep process.

ABSTRACT: The mechanism of substrate translocation through the ribosome is central to the rapid and faithful translation of mRNA into proteins. The rate-limiting step in translocation is an unlocking process that includes the formation of an "unlocked" intermediate state, which requires the convergence of large-scale conformational events within the ribosome including tRNA hybrid states formation, closure of the ribosomal L1 stalk domain, and subunit ratcheting. Here, by imaging of the pretranslocation ribosome complex from multiple structural perspectives using two- and three-color single-molecule fluorescence resonance energy transfer, we observe that tRNA hybrid states formation and L1 stalk closure, events central to the unlocking mechanism, are not tightly coupled. These findings reveal that the unlocked state is achieved through a stochastic-multistep process, where the extent of conformational coupling depends on the nature of tRNA substrates. These data suggest that cellular mechanisms affecting the coupling of conformational processes on the ribosome may regulate the process of translation elongation.

SUBMITTER: Munro JB

PROVIDER: S-EPMC2818936 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Spontaneous formation of the unlocked state of the ribosome is a multistep process.

Munro James B JB Altman Roger B RB Tung Chang-Shung CS Cate Jamie H D JH Sanbonmatsu Kevin Y KY Blanchard Scott C SC

Proceedings of the National Academy of Sciences of the United States of America 20091217 2

The mechanism of substrate translocation through the ribosome is central to the rapid and faithful translation of mRNA into proteins. The rate-limiting step in translocation is an unlocking process that includes the formation of an "unlocked" intermediate state, which requires the convergence of large-scale conformational events within the ribosome including tRNA hybrid states formation, closure of the ribosomal L1 stalk domain, and subunit ratcheting. Here, by imaging of the pretranslocation ri ...[more]

PMID: 20018653

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Project description:Biomolecular simulations provide a means for exploring the relationship between flexibility, energetics, structure, and function. With the availability of atomic models from X-ray crystallography and cryoelectron microscopy (cryo-EM), and rapid increases in computing capacity, it is now possible to apply molecular dynamics (MD) simulations to large biomolecular machines, and systematically partition the factors that contribute to function. A large biomolecular complex for which atomic models are available is the ribosome. In the cell, the ribosome reads messenger RNA (mRNA) in order to synthesize proteins. During this essential process, the ribosome undergoes a wide range of conformational rearrangements. One of the most poorly understood transitions is translocation: the process by which transfer RNA (tRNA) molecules move between binding sites inside of the ribosome. The first step of translocation is the adoption of a "hybrid" configuration by the tRNAs, which is accompanied by large-scale rotations in the ribosomal subunits. To illuminate the relationship between these rearrangements, we apply MD simulations using a multi-basin structure-based (SMOG) model, together with targeted molecular dynamics protocols. From 120 simulated transitions, we demonstrate the viability of a particular route during P/E hybrid-state formation, where there is asynchronous movement along rotation and tRNA coordinates. These simulations not only suggest an ordering of events, but they highlight atomic interactions that may influence the kinetics of hybrid-state formation. From these simulations, we also identify steric features (H74 and surrounding residues) encountered during the hybrid transition, and observe that flexibility of the single-stranded 3'-CCA tail is essential for it to reach the endpoint. Together, these simulations provide a set of structural and energetic signatures that suggest strategies for modulating the physical-chemical properties of protein synthesis by the ribosome.

Dataset Information

Spontaneous formation of the unlocked state of the ribosome is a multistep process.

Publications

Spontaneous formation of the unlocked state of the ribosome is a multistep process.

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