A new view of protein synthesis: mapping the free energy landscape of the ribosome using single-molecule FRET - PubMed (original) (raw)

Review

A new view of protein synthesis: mapping the free energy landscape of the ribosome using single-molecule FRET

James B Munro et al. Biopolymers. 2008 Jul.

Abstract

This article reviews the application of single-molecule fluorescence resonance energy transfer (smFRET) methods to the study of protein synthesis catalyzed by the ribosome. smFRET is a powerful new technique that can be used to investigate dynamic processes within enzymes spanning many orders of magnitude. The application of wide-field smFRET imaging methods to the study of dynamic processes in the ribosome offers a new perspective on the mechanism of protein synthesis. Using this technique, the structural and kinetic parameters of tRNA motions within wild-type and specifically mutated ribosome complexes have been obtained that provide valuable new insights into the mechanism and regulation of translation elongation. The results of these studies are discussed in the context of current knowledge of the ribosome mechanism from both structural and biophysical perspectives.

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Figures

FIGURE 1

FIGURE 1

Single-molecule imaging brings static structural models to life. (A) The ribosome is a processive enzyme that moves along mRNA in 3-nucleotide steps. In vivo, many ribosomes may translate the same mRNA concurrently, forming a polysome (Electron micrograph from nobelprize.org). (B) High resolution structural models of the ribosome provide detailed visualizations of the protein synthesis machinery. (C) Single-molecule methods aim at understanding how dynamic processes of tRNA within the A and P sites of the ribosome relate to the mechanism of translocation.

FIGURE 2

FIGURE 2

A schematic representation of how the ribosome energy landscape may be modulated to promote translocation. In this highly-simplified view, each curve schematically represents the energy landscape of the ribosome at discrete steps in translocation. The free energy of the system is shown as a function of the P-site tRNA position on the ribosome before EF-G binding (_t_0), after EF-G binding (_t_1) and after translocation (_t_2). This schematic is meant to portray only how the global minimum of the system may evolve to promote forward progression. The absolute height of the energy barriers depicted at _t_0, _t_1, and _t_2 are not drawn to scale and should not be compared to one another. Curves are off-set on the _Y_-axis for visualization purposes. Arrows linking each of the three states are shown to indicate multiple steps may separate _t_0, _t_1, and _t_2.

FIGURE 3

FIGURE 3

Wide-field, single-molecule FRET measurements provide direct observations of conformational dynamics of tRNAs on individual ribosomes. Anticorrelated changes in donor (green) and acceptor (red) fluorescence of dyes linked to tRNA report on the dynamics of tRNA within the ribosome. The efficiency of FRET (blue) reports on the distance separating the two dyes. Hidden Markov modeling, resulting in the idealization of the FRET trajectories (overlaid in red), is used to identify the FRET value and lifetimes of each FRET state observed as well as the order of transitions in the system. Here, FRET trajectories have been idealized to a model containing 3 states corresponding to the classical state (C), hybrid state 1 (H1), and hybrid state 2 (H2). (A) tRNAs on the wild-type ribosome are found to occupy predominantly a high-FRET classical configuration with transient excursions to the lower FRET hybrid states. (B) Mutation of residue G2553 of the A loop, and (C) G2252 of the P loop dramatically alter the stability of tRNA configurations on the ribosome, changing the energy landscape to favor intermediate- (B) and low- (C) FRET hybrid states.

FIGURE 4

FIGURE 4

Mutations in key ribosome-tRNA interaction sites aided in identifying and stabilizing hybrid state intermediates on the ribosome. The 3′-CCA termini of A and P site tRNA make critical contacts with the A and P loops of 23S rRNA in the peptidyl transferase center of the 50S subunit.– (A) Here the CCA termini shown in red, of the A- (right) and P- (left) site tRNA are shown in a surface-rendered crystal structure of the peptidyl transferase center. rRNA is shown in tan, with the A and P loops highlighted in brown. Protein components (mainly L27) are shown in blue. (B) Mutations G2553C in the A loop and G2252C in the P loop interrupted base-pairing interactions between C75 and C74 of the A- and P-site tRNA, respectively. Mutation in the A loop promoted formation of the A/P hybrid state, giving rise to an A/P-P/E hybrid state configuration. The P loop mutation promoted formation of the previously unidentified A/A-P/E hybrid state, which leave the 50S P site vacant.

FIGURE 5

FIGURE 5

Single-molecule FRET studies resulted in a new model of tRNA dynamics on the ribosome. The large activation energies associated with transitions between tRNA hybrid states indicate that these transitions are coupled to large-scale conformational rearrangements of the ribosome. A key observation was that tRNA can move independently on the ribosome. The motions of each tRNA appear to be governed by distinct conformational changes of the ribosome as indicated by the coloration of the ribosomal domains.

FIGURE 6

FIGURE 6

Transition density plots depict the remodeling of the free energy landscape due to mutation in tRNA binding sites. Plotting the initial and final FRET values of each transition indicates the existence of three distinct FRET states corresponding to the classical state and two hybrid states. The distribution of transitions shifts as a result of alterations in the energy landscape of the ribosome-tRNA complex due to removal of ribosomal protein L1 (A), or mutations made in key rRNA residues (B–D).

FIGURE 7

FIGURE 7

MD simulations predict the locations of the fluorophores bound to the A and P site tRNAs. (A) Cy5 bound to the A site tRNAPhe at acp3U47 is shown from the leading edge of the ribosomal A site. (B) The P-site tRNAfMet is shown with Cy3 bound at s4U8 as viewed from the E site. H69 and H38 (A-site finger) are identified as reference points (K. Sanbonmatsu, unpublished data). The structures predict ample space for the fluorophores to reside, consistent with measurements of fluorescence anisotropy.

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