Elongation factor 4 remodels the A-site tRNA on the ribosome

Abstract

During translation, a plethora of protein factors bind to the ribosome and regulate protein synthesis. Many of those factors are guanosine triphosphatases (GTPases), proteins that catalyze the hydrolysis of guanosine 5′-triphosphate (GTP) to promote conformational changes. Despite numerous studies, the function of elongation factor 4 (EF-4/LepA), a highly conserved translational GTPase, has remained elusive. Here, we present the crystal structure at 2.6-Å resolution of the Thermus thermophilus 70S ribosome bound to EF-4 with a nonhydrolyzable GTP analog and A-, P-, and E-site tRNAs. The structure reveals the interactions of EF-4 with the A-site tRNA, including contacts between the C-terminal domain (CTD) of EF-4 and the acceptor helical stem of the tRNA. Remarkably, EF-4 induces a distortion of the A-site tRNA, allowing it to interact simultaneously with EF-4 and the decoding center of the ribosome. The structure provides insights into the tRNA-remodeling function of EF-4 on the ribosome and suggests that the displacement of the CCA-end of the A-site tRNA away from the peptidyl transferase center (PTC) is functionally significant.T ranslation of the genetic information requires protein factors that interact with the ribosome sequentially, regulate its activity, and guide it through the protein synthesis cycle in a concerted manner. Many of those factors are guanosine triphosphatases (GTPases), proteins that use energy from guanosine 5′-triphosphate (GTP) to promote conformational changes that lead to transitions between ribosome functional states (1, 2). In bacteria, for instance, initiation of protein synthesis is largely regulated by initiation factor 2 (IF-2), a GTPase that stabilizes the initiator tRNA in the P site of the ribosome (3). Subsequently, the elongation step is catalyzed by two universally conserved GTPases, elongation factor Tu (EF-Tu) and elongation factor G (EF-G). The ternary complex, consisting of EF-Tu, GTP, and the aminoacyl-tRNA, interacts with the ribosome to decode the codon in the A site of the ribosome. Following accommodation of the aminoacyl-tRNA in the A site of the ribosome and subsequent peptide bond formation, the tRNA-mRNA duplex is translocated by one codon-a process catalyzed by EF-G and GTP (4-6). Termination of protein synthesis is triggered when a stop codon is reached, upon which the newly synthesized protein is released with the help of release factor 3 (RF-3), yet another GTPase (7).Elongation factor 4 (EF-4/LepA) is a highly conserved protein structurally similar to EF-G (8) and has a ribosome-dependent GTPase activity (9-13). However, despite numerous studies, its function has remained elusive (9-20). Fast kinetic studies showed that EF-4 competes with EF-G during elongation for binding to the pretranslocation (PRE) ribosome, with tRNAs in the A and P sites (17). Despite this, EF-4 was also shown to increase the rate of protein synthesis at high intracellular ionic strength (16), without any effect on translational accuracy (16, 18). Conversely, EF-4 was also reported...

EF4 reveals the energy barrier for tRNA back-translocation in the peptidyl transferase center

“…3B). Together with our publication, 7,17,18 , all the EF4-ribosome structures confirm that EF4 stabilizes the PRE complex via its CTD region. The stabilization of PRE complex by EF4 may explain the recent results of smFRET study, which suggests that EF4 preferentially targets ribosome in the PRE instead of POST state.…”

Structure of the GTP Form of Elongation Factor 4 (EF4) Bound to the Ribosome

“…Addendum-While this paper was under revision, a crystal structure of the EF4 in its GTP form bound to the PRE-state ribosome was published (PDB code not released) (37) that is very similar to that in Ref. 19.…”

Conserved GTPase LepA (Elongation Factor 4) functions in biogenesis of the 30S subunit of the 70S ribosome

“…This position of LepA is incompatible with A/A-bound tRNA, as the CTD occupies the 50S A site. In those complexes with three tRNAs bound, the A-tRNA adopts a distorted conformation (termed "A/L-tRNA") to avoid steric clash with bound LepA (42). To our knowledge, there is no evidence that ribosome biogenesis involves A-site tRNA; thus, we doubt that the A/L state holds relevance for this process.…”

“…Several ribosome-bound LepA structures have been reported (25,(42)(43)(44). In all cases, mRNA and tRNAs were also part of the complex, and the data were often interpreted with the assumption that LepA acts during elongation.…”