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.

Fig. 1.

Structural models of locked and unlocked states of the pretranslocation ribosome complex showing estimated distances between fluorescently labeled components. A The locked ribosome configuration consistent with the low-FRET (approximately 0.1) state observed on complexes with labeled L1 and P-site tRNA, and the high-FRET (approximately 0.54) state observed on complexes with labeled A- and P-site tRNAs. B The unlocked ribosome configuration consistent with the high-FRET (approximately 0.65) state observed in complexes with labeled L1 and P-site tRNA, and the intermediate-FRET (approximately 0.4) state observed with both tRNAs labeled. The ribosome is shown in (Gray), A-site tRNA in (Cyan), P-site tRNA in (Blue), mRNA in (Red) and the L1 protein in (Magenta). The estimated center of mass of donor (Green) and acceptor (Red) fluorophores used are shown as semitransparent circles.

Fig. 2.

P/E hybrid state formation and L1 stalk closure are not tightly coupled in pretranslocation complexes containing initiator tRNA in the P-site. The dynamics of the pretranslocation complex containing P-site tRNA^fMet and A-site fMet-Phe-tRNA^Phe are shown from two distinct structural perspectives. A L1- and P-site tRNA-labeled complexes. B A- and P-site tRNA-labeled complexes. Left Cartoon models of the labeling sites in the pretranslocation complex and the putative dynamic elements; Cy5 (Red Star), Cy3 (Green Star). Center Single-molecule fluorescence (Cy3, (Green); Cy5, (Red)) and FRET (Blue) trajectories, where FRET idealization obtained through hidden Markov Modeling methods (Red) is overlaid on the FRET trace. Right Nonzero FRET state occupancies identified through idealization. (Pink) and (Red) shaded distributions correspond to those states representing P/E hybrid tRNA configurations and the unlocked state of the ribosome, respectively.

Fig. 3.

Stabilization of the P/E hybrid state does not substantially increase unlocked state occupancy. Data from pretranslocation complex containing P-site tRNA^fMet and A-site fMet-Phe-tRNA^Phe, in the context of a G2252C mutation in the P loop of 23S rRNA are shown from two distinct structural perspectives. A L1- and P-site tRNA-labeled complexes. B A- and P-site tRNA-labeled complexes.

Fig. 4.

P/E hybrid state formation and L1 stalk closure are not tightly coupled in pretranslocation complexes containing initiator tRNA in the P-site. Data from pretranslocation complex containing P-site tRNA^Phe and A-site NAc-Phe-Lys-tRNA^Lys are shown from two distinct structural perspectives. A L1- and P-site tRNA-labeled complexes. B A- and P-site tRNA-labeled complexes.

Fig. 5.

Uncoupled motions of P-site tRNA and the L1 stalk observed through multicolor smFRET. smFRET trajectories acquired from pretranslocation complexes bearing Cy5.5-labeled L1, Cy5-labeled A-site tRNA, and Cy3-labeled P-site tRNA. Data is shown for complexes containing A P-site tRNA^fMet and A-site fMet-Phe-tRNA^Phe, or B P-site tRNA^Phe and A-site NAc-Phe-Lys-tRNA^Lys. From Top to Bottom, the first three panels show Cy3/Cy5, Cy3/Cy5.5, Cy5/Cy5.5 fluorescence trajectories from a single complex. Bottom panels, and insets, show anticorrelated changes in Cy5- and Cy5.5 FRET channels indicative of FRET between Cy5 and Cy5.5 fluorophores in the unlocked state.