tRNA dynamics on the ribosome during translation

Abstract

Using single-molecule fluorescence spectroscopy, time-resolved conformational changes between fluorescently labeled tRNA have been characterized within surface-immobilized ribosomes proceeding through a complete cycle of translation elongation. Fluorescence resonance energy transfer was used to observe aminoacyl-tRNA (aa-tRNA) stably accommodating into the aminoacyl site (A site) of the ribosome via a multistep, elongation factor-Tu dependent process. Subsequently, tRNA molecules, bound at the peptidyl site and A site, fluctuate between two configurations assigned as classical and hybrid states. The lifetime of classical and hybrid states, measured for complexes carrying aa-tRNA and peptidyl-tRNA at the A site, shows that peptide bond formation decreases the lifetime of the classical-state tRNA configuration by approximately 6-fold. These data suggest that the growing peptide chain plays a role in modulating fluctuations between hybrid and classical states. Single-molecule fluorescence resonance energy transfer was also used to observe aa-tRNA accommodation coupled with elongation factor G-mediated translocation. Dynamic rearrangements in tRNA configuration are also observed subsequent to the translocation reaction. This work underscores the importance of dynamics in ribosome function and demonstrates single-particle enzymology in a system of more than two components.

Fig. 1.

70S ribosome showing binding sites for A-, P-, and E-site tRNA molecules (model coordinates courtesy of M. Yusopov, Laboratoire de Biologie et Genomique Structurales de Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France). The 50S subunit is shown in purple and the 30S subunit in tan. aa-tRNA (A), peptidyl-tRNA (P), and deacylated-tRNA (E) binding sites are labeled in black at their approximate location on the 70S ribosome. A-site tRNA is shown in gold and P-site tRNA in red; mRNA is shown in blue. The locations of the peptidyltransferase center and decoding site are highlighted.

Fig. 2.

Single-molecule puromycin assay. Surface-immobilized ribosome complexes carrying Cy3-Met-tRNA^fMet in the P site (dye-labeled at the α-amino group) react with puromycin. Stopped-flow addition of puromycin (1 mM, pH 7.5) to the immobilized complexes results in peptide bond formation and rapid loss of Cy3 signal caused by dissociation of the puromycin-Met-Cy3 product from the surface. Before A-site tRNA accommodation, the loss of fluorescence was fit to a double exponential, A₁*exp(–t/τ₁) + A₂* exp(–t/τ₂) + y₀, where A₁ and A₂ represent the population (%) of fast and slow decay reactions (solid lines). The fitting parameters from a double-exponential fit were: A₁ = 79, τ₁ = 12.6 ± 0.3 s, A₂ = 18, τ₂ = 236 ± 26 s, and y₀ = 3. After enzymatic delivery of Phe-tRNA^Phe, the rate of puromycin reaction is dramatically reduced, consistent with tRNA accommodation at the A site and peptide bond formation. Less that 5% of the population shows a fast decay after Phe-tRNA^Phe delivery. However, some residual reaction with puromycin remains. For comparison, an independent measurement of the intrinsic Cy3 signal decay caused by peptidyl-tRNA dissociation from the A site is shown (dotted line). Recovery of the rapid puromycin reaction was achieved by incubation with EF-G in the presence of GTP. The fitting parameters were A₁ = 58, τ₁ = 6.4 ± 0.3 s, A₂ = 27, τ₂ = 154 ± 8 s, and y₀ = 15. Thus, ≈73% (58/79) of ribosomes return to reacting rapidly with puromycin.

Fig. 3.

Single-molecule Cy3 and Cy5 fluorescence and FRET. tRNA delivery to ribosome complexes carrying fMet-tRNA^fMet(Cy3-s⁴U) in the P site was monitored under Cy3 excitation. Cy5 fluorescence arises from Phe-tRNA^Phe(Cy5-acp³U) binding to the ribosome and colocalization of Cy3 and Cy5 fluorophores within the same complex. (Upper) Cy3 and Cy5 emission intensity are shown in green and red, respectively. (Lower) The corresponding FRET value, I_Cy5/(I_Cy3 + I_Cy5), is shown in blue.

Fig. 4.

Postsynchronized, time-resolved population FRET histograms reporting on intermolecular tRNA–tRNA distances within individual ribosomes of an ensemble of particles. Histograms show the evolution of FRET for an ensemble of surface-immobilized 70S complexes carrying tRNA^fMet(Cy3-s⁴U) in the P site after delivery of EF-Tu(GTP)Phe-tRNA^Phe(Cy5-acp³U) where individual FRET time traces have been synchronized to the first FRET value ≥ 0.25. Population information is color-coded from tan (lowest occupancy) to red (highest occupancy). The histogram plots generated by stopped-flow delivery of EF-Tu(GTP)Phe-tRNA^Phe(Cy5-acp³U) depend on the acylation state of tRNA in the P site and in the presence of EF-G. (A) Complexes carrying fMet-tRNA^fMet(Cy3-s⁴U) in the P site. (B) Complexes carrying OH-tRNA^fMet(Cy3-s⁴U) in the P site. (C) Exactly as in A but in the presence of EF-G. Within experimental uncertainty, the high FRET peaks observed have the same mean FRET value, 0.74 ± 0.05. Similarly, the lower FRET peaks observed in A and B have a mean FRET value of 0.45 ± 0.05. Peaks centered at zero FRET arise from Cy5 blinking, photobleaching, and tRNA dissociation.