Unraveling the dynamics of ribosome translocation

Abstract

Translocation is one of the key events in translation, requiring large-scale conformational changes in the ribosome, movements of two transfer RNAs (tRNAs) across a distance of more than 20Å, and the coupled movement of the messenger RNA (mRNA) by one codon, completing one cycle of peptide-chain elongation. Translocation is catalyzed by elongation factor G (EF-G in bacteria), which hydrolyzes GTP in the process. However, how the conformational rearrangements of the ribosome actually drive the movements of the tRNAs and how EF-G GTP hydrolysis plays a role in this process are still unclear. Fluorescence methods, both single-molecule and bulk, have provided a dynamic view of translocation, allowing us to follow the different conformational changes of the ribosome in real-time. The application of electron microscopy has revealed new conformational intermediates during translocation and important structural rearrangements in the ribosome that drive tRNA movement, while computational approaches have added quantitative views of the translational pathway. These recent advances shed light on the process of translocation, providing insight on how to resolve the different descriptions of translocation in the current literature.

Figure 1

Single-molecule translation assays (a) Cy5-labeled 50S subunits, tRNA ternary complex, and EF-G are delivered to surface-immobilized single pre-initiation complexes containing Cy3-labeled 30S subunits and illuminated at 532 nm; both Cy3 and Cy5 fluorescence are simultaneously detected. Immobilization with an mRNA coding for six phenylalanines (6F) permits the observation of ribosome conformation during multiple rounds of elongation via the intersubunit FRET signal. The arrival of FRET corresponds to 50S subunit joining during initiation and is followed by multiple cycles of high-low-high FRET, each reporting on ribosome unlocking and locking during one round of elongation, according to Spirin’s model. The unlocked state is rotated, while the locked state is non-rotated. (b) FRET between helix 44 (30S) and helix 101 (50S) reports on the relative rotational states of the 30S body with respect to the 50S subunit. Figure obtained from [50] (c) Single-molecule time traces of S6-Cy5/L9-Cy3 ribosome complexes showing intersubunit rotation of the 30S head domain induced by peptide bond formation (puromycin), EF-G binding, and translocation. The ribosome starts in the high FRET state. Peptide bond formation results in a transition to the low FRET state but also induces spontaneous intersubunit head rotational movements, as indicated but the fluctuations between the low FRET state and high FRET state. EF-G binding stabilizes the low FRET state. Translocation by EF-G is observed as the transition back to the high FRET state. This also echoes Spirin’s unlocking/locking model. (d) Ribosomes labeled at L9 and S6 or S11. FRET reveals spontaneous conformational fluctuations similar to those observed for the L1 stalk and tRNA. Reproduced with modifications and with permission from Aitken et al. and Cornish et al. [31, 35].

Figure 2. Cryo-EM reveals sub-states of translation

(a) Distribution of 30S substates for different tRNA positions in pre and post states. The heat map indicates the fraction of particles relative to the total number of particles in the respective state. The transition from Pre1 to Pre2 (likely peptide bond formation) results in a large 30S body rotation counterclockwise. Up until translocation, the 30S head also rotates. After translocation (from Pre5 to Post1), both the 30S head and 30S body rotates back, resetting the ribosome for the next round of elongation. (b) Schematic of 30S body rotation. The 30S body rotates around a pivot point at helix 27 of 16S rRNA independent of the 30S head. Schematic of 30S head movement. 30S head movement comprises a rotation and swiveling motion around the neck region (h28). This indicates that multiple ribosome conformational changes coordinate translocation. (c) Free energy landscape of global ribosome conformation of the different tRNA substates. Reproduced with modifications and with permission from Fischer et al. [41].

Figure 3. EF-G Dynamics

The conformations of EF-G in various bound states, as observed by both X-ray crystallography and cryo-EM: EF-G•GTP in unbound form (red); EF-G•GTP bound to the ribosome (yellow); EF-G•GDP bound to the ribosome (blue); and EF-G•GDP in unbound form (pink). EF-G conformations changes throughout the elongation cycle. These conformations may be significant in determining binding dynamics of EF-G to the ribosome as well as catalyzing ribosome conformational changes. Reproduced with permission from Li et al. [44].

Figure 4. Schematic of elongation

The ribosome is separated into two global conformations, the “unlocked” state and the “locked” state. Upon peptide bond formation, the ribosome “unlocks” and the 30S subunit rotates with respect to the 50S subunit, permitting fluctuations of the L1 stalk between open and closed states, fluctuations of tRNAs between the classical and hybrid states, and spontaneous rotations in the 30S head domain. EF-G•GTP binding then stabilizes the L1 stalk in the closed state and tRNA in the hybrid state, as well as causing the head of the 30S subunit to rotate. GTP hydrolysis by EF-G unlocks movement of the mRNA for translocation, followed by relocking of the ribosome (back-rotation of the 30S body and head domains) and translocation. Relocking of the mRNA arrests its movement to preserve reading frame. The E-site tRNA and EF-G•GDP departs rapidly, returning the ribosome to the original state.