Allosteric collaboration between elongation factor G and the ribosomal L1 stalk directs tRNA movements during translation

Abstract

Determining the mechanism by which tRNAs rapidly and precisely transit through the ribosomal A, P, and E sites during translation remains a major goal in the study of protein synthesis. Here, we report the real-time dynamics of the L1 stalk, a structural element of the large ribosomal subunit that is implicated in directing tRNA movements during translation. Within pretranslocation ribosomal complexes, the L1 stalk exists in a dynamic equilibrium between open and closed conformations. Binding of elongation factor G (EF-G) shifts this equilibrium toward the closed conformation through one of at least two distinct kinetic mechanisms, where the identity of the P-site tRNA dictates the kinetic route that is taken. Within posttranslocation complexes, L1 stalk dynamics are dependent on the presence and identity of the E-site tRNA. Collectively, our data demonstrate that EF-G and the L1 stalk allosterically collaborate to direct tRNA translocation from the P to the E sites, and suggest a model for the release of E-site tRNA.uring the elongation phase of protein synthesis, the ribosome repetitively cycles through three principal steps: (i) selection of an aminoacyl-transfer RNA (tRNA) at the ribosomal A site (1), (ii) peptidyl transfer from the P site-bound peptidyl-tRNA to the A site-bound aminoacyl-tRNA (2), and (iii) translocation of the messenger RNA (mRNA)-tRNA complex by one codon, effectively moving the P-and A-site tRNAs into the E-and P-sites, respectively (3). Perhaps the most dynamic steps of this cycle are the precisely directed mRNA and tRNA movements that occur during translocation (3-5). Although this step of the elongation cycle is promoted by elongation factor G (EF-G), numerous biochemical (6), structural (7,8), and Förster resonance energy transfer (FRET) (9-15) studies have provided strong evidence that the peptidyl transfer step of the elongation cycle spontaneously triggers an EF-Gindependent structural rearrangement of the ribosomal pretranslocation (PRE) complex that involves movements of the ribosome-bound tRNAs from their classical to their hybridbound configurations (6-10, 12), movement of the ribosomal L1 stalk from an open to a closed conformation (7,8,13,15), and a counterclockwise, ratchet-like rotation of the small ribosomal subunit relative to the large subunit (7,8,11,14).Single-molecule FRET (smFRET) investigations have proven a powerful means for directly investigating the conformational dynamics of PRE complexes. Aided by X-ray and cryogenic electron microscopy (cryo-EM)-derived structural models, several groups have reported kinetic studies of tRNA and ribosome movements within PRE complexes (9, 10, 12-15). tRNA-tRNA smFRET (smFRET tRNA-tRNA ) experiments initially revealed that upon peptidyl transfer, tRNAs enter into a classicalĥ ybrid dynamic equilibrium within PRE complexes (9, 10, 12). More recently, we have used an L1 stalk-tRNA smFRET (smFRET L1-tRNA ) signal to demonstrate that upon peptidyl transfer, a direct L1 stalk-tRNA contact is reversibly established (denoted as L1YtR...

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

“…2 and Fig. S1A) (14). Likewise consistent with previous studies, the smFRET L1-tRNA signals from all PRE -A complexes exhibited fluctuations between a FRET state with an E FRET = 0.15 ± 0.02, assigned to the L1•tRNA conformation of the PRE -A complex, and a FRET state with an E FRET = 0.74 ± 0.03, assigned to the the L1•tRNA conformation of the PRE -A complex, thus reporting on the L1•tRNA⇆L1•tRNA equilibrium ( Fig.…”

“…L1 stalk and tRNA dynamics of PRE/PRE -A complexes can be monitored using two previously reported smFRET signals. The first signal, denoted smFRET L1-L9, derives from Cy3 FRET donor-labeled r-protein L9 [L9(Cy3)] and Cy5 FRET acceptorlabeled r-protein L1 [L1(Cy5)] and reports on movements of the L1 stalk between its L1 o and L1 c conformations (14). The second signal, denoted smFRET L1-tRNA , derives from Cy3-labeled P-site tRNA [tRNA(Cy3)] and L1(Cy5) and reports on rearrangements of the PRE/PRE -A complex between a conformation in which the L1 stalk is in its L1 o conformation and is too far away to physically interact with the P/P-configured tRNA (L1•tRNA) and a conformation in which the L1 stalk is in its L1 c conformation and is able to physically interact with the P/E-configured tRNA (L1•tRNA) (21).…”

“…Previously, we (14) and others (15) have shown that in addition to stabilizing the R-subunit orientation (13), binding of EF-G to PRE -A complexes stabilizes the L1 c conformation of the L1 stalk. This occurs despite the fact that EF-G does not directly contact either the L1 stalk or the intervening P-site tRNA within the PRE -A complex (14).…”

“…Our findings suggest that such coordination is likely to be a general and important mechanism through which all biomolecular machines maximize and regulate their functional efficiencies. PRE complex analogs lacking an A-site tRNA (PRE -A complexes) undergo thermally activated, stochastic, and reversible fluctuations between classical and hybrid tRNA configurations (classical⇆hybrid) (12), nonrotated and rotated subunit orientations (NR⇆R) (13), and open and closed L1 stalk conformations (L1 o ⇆L1 c ) (14,15). Unfortunately, however, whether and how these stochastic tRNA, intersubunit, and L1 stalk dynamics are coordinated within PRE/PRE -A complexes to facilitate translocation and/or allosteric regulation of translocation remains unknown, severely limiting our understanding of the fundamental physical processes that drive and control the rapid and precise translocation of the tRNAs through the ribosome during protein synthesis.…”

Selenocysteine Insertion Sequence (SECIS)-binding Protein 2 Alters Conformational Dynamics of Residues Involved in tRNA Accommodation in 80 S Ribosomes

“…This intersubunit bridge was also observed in the structure of the mammalian 80 S ribosome obtained by cryoelectron microscopy (30). The L1 stalk has been implicated in the movement and release of deacylated tRNAs from the E-site (31,32). In addition, E-site clearance is allosterically modulated by the tRNA status of the A-site (33).…”