The ribosome uses cooperative conformational changes to maximize and regulate the efficiency of translation

Abstract

One of the most challenging unanswered questions regarding the structural biology of biomolecular machines such as the two-subunit ribosome is whether and how these machines coordinate seemingly independent and random conformational fluctuations to maximize and regulate their functional efficiencies. To address this question, we have used ribosome mutagenesis or a ribosome-targeting antibiotic to predictably perturb the dynamics of intersubunit rotation, a structural rearrangement of the ribosome that is essential for the translocation and ejection of ribosome-bound tRNAs during translation. Concomitantly, we have used single-molecule fluorescence resonance energy transfer (smFRET) to characterize the effects of these perturbations on the dynamics of ribosomal L1 stalk movements and ribosome-bound tRNA reconfigurations, conformational changes that are likewise essential for the translocation and ejection of tRNAs during translation. Together with the results of complementary biochemical studies, our smFRET studies demonstrate that the ribosome uses cooperative conformational changes to maximize and regulate the efficiency with which it translocates and ejects tRNAs during translation. We propose that the ribosome employs cooperative conformational changes to efficiently populate global conformational states that are productive for translation, that translation factors exploit this cooperativity as part of their mechanisms of action, and that antibiotics exploit it to maximize the potency with which they inhibit translation. It is likely that similar cooperative conformational changes underlie the function and regulation of other biomolecular machines. During the catalytic cycle of many enzymes, multiple, spatially distant enzyme structural elements must often undergo functionally important conformational changes (1). Consistent with the view that such structural rearrangements must be rapidly organized and executed to maintain catalytic efficiency, many recent studies strongly suggest that small, monomeric protein enzymes have evolved complex networks of cooperative conformational changes that coordinate the inherently stochastic conformational fluctuations of multiple structural elements in a manner that is optimal for catalysis (2). Within the context of energy landscape theory (3), such enzymes can be thought of as having evolved dynamic energy landscapes that bias enzyme conformational sampling in such a manner to maximize the efficiency of catalysis (2). Related proposals suggest that binding of allosteric effectors to enzymes remodels such energy landscapes, altering enzyme conformational sampling as part of the mechanisms through which these effectors regulate enzymatic activity (4).Unfortunately, the conformational dynamics of large, macromolecular complexes remain much more challenging to characterize than those of small, monomeric proteins (5), making it very difficult to elucidate the role that cooperative conformational changes play in the function and regulation of biomolecular machines such as the ...

Initiation factor 2 stabilizes the ribosome in a semirotated conformation

“…Therefore, coupling between the formation of the half-closed conformation of the L1 stalk and IF2-induced stabilization of the semirotated conformation of the ribosome is likely not mediated by the interaction between the L1 stalk and tRNA bound either in the P/I or E/E state. Consistent with our results, recent smFRET data showed that coupling between intersubunit rotation and L1 stalk movement can occur in vacant ribosomes (36), further supporting the idea that the inward movement of the L1 stalk does not require interaction between the L1 stalk and tRNA.…”

Two promising future developments of cryo-EM: capturing short-lived states and mapping a continuum of states of a macromolecule

“…This series of observation in Chen et al [30] is in good agreement with the results of a fast chemical probing assay [41] and a number of single-molecular fluorescence resonance energy transfer studies [42][43][44], but disagrees with another chemical probing assay [45] and the previous timeresolved cryo-EM study [29]. In comparing the two timeresolved cryo-EM studies, the major reason why the two studies reached different conclusions is the difference in the resolutions of maps (9-12 Å in Chen et al [30] and 23-33 Å in Shaikh et al [29]), which stemmed from the difference in data yield.…”

Molecular mechanism of viomycin inhibition of peptide elongation in bacteria

“…This suggests that A-site binding of a tRNA liberates the viomycin binding site, which would otherwise be occupied by the monitoring bases. Further evidence that A-site binding of tRNA increases viomycin affinity to the ribosome comes from data presented in a recent single-molecule study (13). From their data we estimate a dissociation constant for viomycin binding to 70S ribosomes with an empty A site as large as 20 μM.…”

“…Translocation is catalyzed by elongation factor G (EF-G) in a GTP hydrolysis-dependent manner (7) and occurs via formation of tRNA hybrid states (8), relative rotation of the ribosomal subunits (9), and movement of the L1 stalk (10). Bulk FRET, chemical footprinting, and single-molecule FRET experiments have shown that viomycin stabilizes the ribosome in a pretranslocation state with tRNAs in hybrid A/P and P/E configurations, rotated ribosomal subunits, and the L1 stalk in a closed conformation interacting with the P-site tRNA (11)(12)(13)(14)(15)(16); this structure has been visualized by cryo-EM (17). However, a crystal structure of the viomycin-bound ribosome (18) and one single-molecule FRET study (19) have suggested stabilization of the tRNAs in the classical state.…”