Identification of two distinct hybrid state intermediates on the ribosome

Abstract

High spatial and time resolution single-molecule fluorescence resonance energy transfer measurements have been used to probe the structural and kinetic parameters of transfer RNA (tRNA) movements within the aminoacyl (A) and peptidyl (P) sites of the ribosome. Our investigation of tRNA motions, quantified on wild-type, mutant, and L1-depleted ribosome complexes, reveals a dynamic exchange between three metastable tRNA configurations, one of which is a previously unidentified hybrid state in which only deacylated-tRNA adopts its hybrid (P/E) configuration. This new dynamic information suggests a framework in which the formation of intermediate states in the translocation process is achieved through global conformational rearrangements of the ribosome particle.

Figure 1. Single-molecule FRET trajectories show three tRNA configurations on the ribosome

(A) Fluorescence and FRET trajectories reveal the existence of three distinct FRET states as indicated. Cy3 and Cy5 fluorescence are shown in green and red, respectively. FRET efficiency (FRET=I_Cy5/(I_Cy3 + I_Cy5)) is shown in blue. The idealization of the data is overlaid in red on the FRET trace. (B) The boxed region in (A) is expanded to highlight specific FRET transitions.

Figure 2. Increasing peptide length increases hybrid state occupancy

(A) Single-molecule FRET trajectories were summed into histograms to reveal the population behavior of complexes bearing deacylated-tRNA^fMet in the P site and (left) Phe-tRNA^Phe, (center) Met-Phe-tRNA^Phe, or (right) fMet-Phe-tRNA^Phe in the A site. Scales shown at the top of each panel of histograms indicate the relative populations. Zero-FRET states arise from both blinking and photobleaching. (B) 1-dimensional histograms of the population data were fit to the sum (black line) of four single Gaussian distributions (red) to estimate the mean values and relative occupancies of each FRET state. Histograms overlaid in blue represent simulated data. (C) Initial and final FRET values for each transition are summed into 2-dimensional histograms and show that transitions occur among distinct FRET states. (D) Histograms generated by the simulation of single-molecule FRET data.

Figure 3. Mutation of A, P, and E sites reveal the physical nature of tRNA binding sites

Data are displayed as in Figure 2. (far-left) L1-depleted complexes; (center-left) wild-type complexes; (center-right) G2553C-mutated complexes; (far-right) G2252C-mutated complexes.

Figure 4. Puromycin reactivity confirms the assignment of FRET states to distinct tRNA configurations on the ribosome

A single-molecule puromycin assay was used to report on the position of the peptide within wild-type, G2552C-mutated, and G2553-mutated ribosome complexes bearing Cy3-Met-tRNA^fMet in the A site. Reaction with puromycin correlates with the occupancy of an A/P hybrid state. Puromycin (2mM) was stopped-flow delivered to surface-immobilized complexes and reaction progress was followed as the loss of fluorescence over time. After consideration of the rates of Cy3 photobleaching, and peptidyl-tRNA drop off from the A site (see discussion Supplemental Figure 3), the reactivities of the three complexes were: wild-type (red squares), k₁=0.081/sec; G2252C (blue circles), k₁=0.036/sec; and G2553C (green triangles), k₁=0.53/sec. In agreement with the predicted positions of the peptide moiety, the rates of puromycin reaction are faster for A-loop mutant ribosomes and slower for P-loop mutant ribosomes than is observed for wild-type pre-translocation complexes.

Figure 5. Equilibrium model of tRNA motions on the ribosome

The two subunit ribosome (yellow) is shown bound to tRNA (blue) and mRNA (green). The three states observed, classified as classical, hybrid state-1, and hybrid state-2, are defined by the motions of tRNA acceptor stems with respect to the large subunit. The apparent rates of transition between states are identified as tabulated in Table 1 and Supplemental Table 3. Conformational changes of the ribosome that may be important to tRNA motions, as described in the text, are shown as changes in A, P, and E site color.