Unraveling the dynamics of ribosome translocation

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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 20 Å, 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.

Highlights

► Translocation requires large-scale ribosome conformational changes, tRNA movement into adjacent sites, and mRNA movement by one codon. ► How the conformational rearrangements of the ribosome are linked to translocation is still unclear. ► Multiple intersubunit rotations and conformational sub-states drive the movement of tRNA and mRNA. ► Additional experiments using cryo-electron microscopy, single-molecule fluorescence, and molecular dynamics will provide further insights into the mechanism of translocation.

Introduction

Protein synthesis requires coordination between the ribosome and multiple translation factors to convert the genetic information encoded by the messenger RNA (mRNA) to a polypeptide sequence [1]. The ribosome is a 2.4 MDa RNA-protein enzyme comprised of two subunits (30S and 50S in prokaryotes) [2, 3, 4]. After successful initiation, that is factor-guided 70S assembly of the two subunits [5, 6], the ribosome commits to the elongation phase. In each cycle of elongation, the ribosome selects the correct aminoacyl transfer RNA (tRNA) specified by the mRNA codon to the aminoacyl-tRNA binding site (A site) [7, 8]. Formation of a peptide bond with the peptidyl-tRNA in the adjacent P site transfers the elongating polypeptide from the P-site tRNA to the A-site tRNA. During the translocation step, catalyzed by elongation factor EF-G [9], the A- and P-site tRNAs must be moved by distances of 20 Å or more to the P site and E site (exit site) respectively [10], accompanied by the movement of the mRNA by precisely one codon [11] with respect to the ribosome to maintain the correct reading frame. The E-site tRNA then dissociates spontaneously [1], leaving the ribosome with a vacant A site and E site, ready for the next round of elongation.

After peptide bond formation ribosome is capable of slowly undergoing spontaneous translocation (at ∼3 h−1)[12]. However, EF-G greatly accelerates the process. The overall rate of EF-G dependent translocation in vitro was measured in bulk to be approximately 20 s−1 at 1 μM EF-G [9, 13]. This matches well with the rate of elongation in vivo, which is found to be ∼20 amino acids per second [14]. After translocation, EF-G dissociates within 50 ms, resetting the ribosome for another round of elongation. These rates frame the timescale for molecular events in translocation.

Determining the molecular mechanism of translocation and open reading-frame maintenance remains one of the key problems in translation. More than four decades ago, a model was proposed by Spirin in which ribosome translocation is controlled by a series of ‘locking’ and ‘unlocking’ events that separate two global conformational states correspondingly termed ‘unlocked’ and ‘locked’ [15]. In this original model, the ribosomal subunits are tightly associated before peptide bond formation to facilitate manipulation of the tRNA and preserve reading frame on the mRNA. After peptide bond formation, the ribosome unlocks. In this unlocked state, the ribosomal subunits and tRNA can move more freely, facilitating translocation of the tRNA and stepping to the next codon of the mRNA [8, 16]. Full translocation returns the ribosome to the locked state, once again restricting the motion of the ribosomal subunits and tRNA.

Consistent with this model, cryoelectron microscopy (cryo-EM) and X-ray crystallography structures revealed that the two subunits of the ribosome undergo dramatic conformational changes after peptide bond formation, by which the small (30S) subunit is rotated ∼3–10° counter clockwise with respect to the large (50S) subunit [17, 18]. This rotational movement is possible because the intersubunit contacts, which consist mainly of RNA–RNA interactions, are relatively labile and can rearrange with little energy cost [18, 19, 20, 21]. However, it is still unknown how these intersubunit rotations relate to Spirin's locking and unlocking mechanism. In addition to the intersubunit rotation, there is also a nearly orthogonal rotation of the head domain of the 30S subunit that seems to play a role in controlling the position of tRNAs within the ribosome [20]. There is thus more than one rotational movement of the ribosome. Recent crystallographic structures of the 70S ribosomal particle have revealed multiple conformational intermediates [22], and here we call the collection of these stepwise conformational changes leading to translocation ‘ratcheting.’ Aside from these global rotational movements of the ribosome, multiple local conformational rearrangements also occur during the various steps of elongation. For example, the L1 stalk is thought to facilitate the movement of tRNA from the P site to the E site [23, 24•], fluctuating between three distinct conformational states.

Upon peptide bond formation, not only does the ribosome itself undergo dramatic conformational changes before translocation, the tRNAs also fluctuate between multiple states. tRNAs can fluctuate freely between the classical (A/A and P/P) state and the hybrid state (A/P and P/E), facilitating the upcoming translocation step catalyzed by EF-G [8, 25, 26, 27]. These conformational changes are possibly driven by one of the many ribosome conformational rearrangements. Chemical probing, subsequent cryo-EM structures, and single-molecule techniques [7, 8] all identified a hybrid tRNA configuration, in which the acceptor stems of the A- and P-site tRNAs interact with the P and E sites of the large subunit, respectively, while the anticodon stem loops of the tRNAs remain in the A and P sites of the small subunit. There have been reports of additional hybrid-state intermediates [28]. This hybrid tRNA configuration can be seen as an intermediate step in translocation. The fluctuation of tRNAs upon peptide bond formation echoes the ‘unlocking/locking’ model proposed by Spirin.

Conformational rearrangements of the ribosome are probably coupled to the tRNA fluctuations and the eventual translocation of the tRNAs and mRNA. Although it is generally assumed that mRNA motion and tRNA movement are coupled mechanically and temporally, this assumption has never been validated directly [22, 25]. However the link between intersubunit rotation and translocation has been established. If two subunits are cross-linked such that the intersubunit rotational movement is not possible, translocation does not occur [29]. While ribosome can undergo factor free translocation, EF-G and GTP hydrolysis by EF-G greatly accelerates the rate of translocation (see above) possibly by driving one of these conformational changes. Determining molecular mechanisms that link ribosome conformation and ligand dynamics to EF-G-catalyzed translocation remains a key question in translation.

In this review, we address recent advances in understanding the mechanism of translocation through dynamic studies by fluorescence methods, structural results by cryo-electron microscopy and X-ray crystallography, and computation by molecular dynamics. We incorporate these recent results with the current views to formulate a consistent model of translocation.

Section snippets

Real-time dynamics of translocation

Fluorescence approaches, either through single-molecule methods or stopped-flow techniques, probe the dynamics of biological processes with high sensitivity and time resolution. Moreover, fluorescence resonance energy transfer (FRET) provides a method to measure dynamic changes in macromolecular conformation by probing the distance (or potentially the orientation) between a donor dye and acceptor dye (usually separated by 20–80 Å)[30]. With single-molecule fluorescence, dynamics can be observed

Structural insights into translocation

Despite the power of fluorescence methods to illuminate dynamics, they provide sparse structural information. Cryo-electron microscopy has been used extensively in structural studies of the ribosome, and acts as a potent complement to FRET. Time-resolved and multiparticlecryo-electron microscopy (cryo-EM) have revealed of how structural rearrangements on different length scales act concertedly to facilitate mechanical processes central to translation, such as translocation. Despite its

Linking structural and dynamic information

Structural snapshots from X-ray crystallography and cryo-EM suggest that many conformational rearrangements of the ribosome, tRNAs, and EF-G must occur during translocation. Nevertheless, high-resolution structures of the transition states in between those snapshots are not available, due to the intrinsic high energy and short lifetimes of these states. Cryo-EM imaging techniques have been successful in capturing some of the intermediate states, albeit at lower resolution and sometimes with

Discussion

Recent results in fluorescence assays, structural studies, and molecular dynamics simulations have shed additional light onto the mechanisms of translocation. However, the current literature in translocation is still marred with confusing and seemingly contradictory models. This problem mostly stems from the misuse of terminology. The terms ‘ratcheting’, ‘unlocking and locking,’ and ‘rotated and non-rotated’ have been used repeatedly in literature (including by us), although each of the studies

Conclusions

Moore once described the current literature on translocation as a ‘proverbial group of blind men trying to describe the proverbial elephant on the basis of what each learns about the elephant by groping it at random’ [49]. This description is not far from the truth. Every group uses different methods to probe translocation, be it cryo-EM, single-molecule FRET, bulk fluorescence methods, or molecular dynamics modeling, they each shed light on different aspects of the process. Thus, further

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

Single-molecule research in the Puglisi group is funded by NIH grants GM51266 and GM099687. We would like to thank all members of Puglisi laboratory for helpful discussions.

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