Navigating the ribosome's metastable energy landscape

Abstract

The molecular mechanisms by which tRNA molecules enter and transit the ribosome during mRNA translation remains elusive. However, recent genetic, biochemical and structural studies offer important new findings into the ordered sequence of events underpinning the translocation process that help place the molecular mechanism within reach. In particular, new structural and kinetic insights have been obtained regarding tRNA movements through 'hybrid state' configurations. These dynamic views reveal that the macromolecular ribosome particle, like many smaller proteins, has an intrinsic capacity to reversibly sample an ensemble of similarly stable native states. Such perspectives suggest that substrates, factors and environmental cues contribute to translation regulation by helping the dynamic system navigate through a highly complex and metastable energy landscape.

Figure I

Schematic structural representation of the four principal phases of translation. This highly simplified diagram shows initiation, elongation, termination and recycling steps, as well as the key translation factors facilitating these reactions, in a manner meant to illustrate the cyclical nature and estimated timescales of translation processes. The range of time scales shown depends strongly on mRNA codon sequence and the availability of substrates and factors, as well as temperature and ionic conditions. The translocation component of the elongation cycle is highlighted in red to indicate the focus on this multistep process in this review.

Figure I

Schematic of ligand-induced modulation of the free-energy landscape. (a) Graph indicating the hierarchical structure of the free energy landscape with the magnitude of activation barriers relative to k_BT (k_B is Boltzmann’s constant, T is temperature). Also shown is the remodeling of the landscape before (black) and after (red) ligand binding. (b) Hypothetical graph diagram indicating the existence of multiple pathways connecting states on the energy landscape and the re-weighting of preferred pathways before (black) and after (red) ligand binding.

Figure 1

Structural snapshots of functional ribosome complexes reveal distinct conformational degrees of freedom implicated in the translation mechanism. Distinct views of three-dimensional, atomic models of the bacterial ribosome (Protein Data Bank accession codes 2QAL, 2QAM, 1VS9 and 1Q86) are shown to schematically illustrate (with double-headed arrows) mobile structural elements within (a) the 70S particle, (b) the large (50S) ribosomal subunit and (c) the small (30S) ribosomal subunit. Ribosomal proteins are shown in blue and tan in the large and small subunit, respectively. In both subunits rRNA is shown in gray ribbons. Conserved rRNA elements directly contacting tRNA substrates are shown in green. (a) A cross-sectional view of the 70S pre-translocation ribosome reveals the substrate-binding channel formed at the subunit interface. The three tRNA binding sites (E, P and A) are illustrated, and classically configured A- and P-site tRNAs are shown in red. Landmark structural features, including the decoding site (DS), the peptidyl transferase center (PTC), the L1 stalk, the GTPase activating center (GAC), the A-site finger (ASF; H38 of 23S rRNA), the P-site gate (G1338–A1339, A790 of 16S rRNA) and the mRNA track are indicated for structural reference. (b) The isolated 50S subunit interface is shown to schematically illustrate the protein and rRNA components of the L1 stalk and the GAC and their estimated range of motions [26,55,57]. The ASF bridging the large and small subunit head domains, as well as the A- and P-loop structural elements within the PTC that base pair with the 3′-CCA-ends of tRNA, are highlighted in green. (c) The solvent surface of the 30S subunit is shown to illustrate the single-stranded mRNA (red) track, which wraps around the neck domain and contacts the Shine–Dalgarno (SD) sequence at the convergence of the three principal structural domains (head, platform and body). Panels (i–iii) show the identified conformational degrees of freedom in the ribosome that are implicated in the translocation mechanism and their approximate magnitudes. (i) Subunit ratcheting rotates the three 30S domains counterclockwise in a collective fashion; the reverse motion is termed unratcheting. (ii) Head rotation represents a swiveling motion of the 30S head domain around the neck-like feature connecting the principal domains [3]. (iii) Head tilt represents flexion of the neck perpendicular and parallel to the subunit interface [35]. (ii–iii) The head domain is highlighted to indicate that these motions can largely occur in the absence of subunit ratcheting.

Figure 2

The multistep process of translocation is one of motion, including conformational changes in the ribosome, tRNA and EF-G. A mechanistic model depicting the process of tRNA–mRNA translocation is schematically diagramed to include data from kinetic, structural and smFRET investigations. Dynamic processes and factor- and substrate-induced conformational changes are included where knowledge of such processes is either known or inferred from experimental data. (i) On the dynamic pre-translocation complex, A- and P-site tRNAs (red) spontaneously transition to the A/P and P/E hybrid states [17,41,42]. The 30S subunit exchanges between ratcheted and unratcheted states [53,58,59], and the L1 stalk transitions between open (opaque), intermediate (translucent) and closed (translucent) positions [56,57]. (Step 1) EF-G binds rapidly and reversibly to the dynamic pre-translocation complex (ii). (Step 2) EF-G binding is quickly followed by GTP hydrolysis (iii) [44,45,80]. (Step 3) GTP hydrolysis promotes subunit ratcheting (RSR), P/E-hybrid-state formation and L1 stalk closure (iv), quickly followed by (Step 4) formation of the A/P hybrid state (v). (Step 5) Rate-limiting conformational changes within the ribosome and EF-G, triggered by the direct interaction of EF-G’s tRNA like domains with the 30S decoding site, precipitate movements of both A- and P-site tRNA–mRNA complexes with respect to the 30S subunit and P_i release (perhaps in random order), followed by unratcheting (vi). (Step 6) EF-G(GDP) releases from the post-translocation ribosome complex. In the post-translocation complex the L1 stalk adopts a distinct, partially closed configuration (vii). Rate constants, where indicated, were compiled from previously published models taken under a variety of experimental conditions and must be interpreted with the understanding that global translocation rates are highly sensitive to both temperature and buffer components as well as ribosome pre-translocation complex composition [8,44,45,50,57]; rates that have not been experimentally determined are noted with a ‘?’. Stochastic processes (tRNA and L1 stalk motions, subunit ratcheting/ unratcheting) are presumed to remain dynamic throughout the process, where prior to translocation only the relative populations of potential sub-states change.

Figure 3

Atomic and cartoon models of classical and hybrid tRNA configurations. (a) Cartoon depicting the transitions of A- and P-site tRNAs (red) between the classical state (A/A–P/P), hybrid state 1 (A/P–P/E) and hybrid state 2 (A/A–P/E) observed by smFRET [41]. The Cy3 and Cy5 fluorophores used in smFRET investigations of tRNA motions attached to naturally occurring modified nucleotides (s⁴U8 on P-site tRNA^fMet and acp³U47 on A-site peptidyl-tRNA^Phe) are represented as green and red circles, respectively. Also shown are the approximate occupancies of the three configurations, the transition rates and the estimated distance changes observed. (b) Atomic models of the classical (A/A and P/P) and hybrid (A/P and P/E) tRNA configurations observed by cryo-EM [59] embedded in an atomic model of the 70S ribosome. As in (a), the approximate positions of the Cy3 (green) and Cy5 (red) fluorophores used in smFRET investigations are shown near the elbow regions of the A- and P-site tRNAs [41]. The distances shown are the estimated inter-dye displacements between classical and hybrid states [59]. Panel (b) was reproduced, with permission, from Ref. [59].