Cryo-EM structure of the mammalian eukaryotic release factor eRF1–eRF3-associated termination complex

Abstract

Eukaryotic translation termination results from the complex functional interplay between two eukaryotic release factors, eRF1 and eRF3, and the ribosome, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl-tRNA hydrolysis by eRF1. Here, using cryo-electron microscopy (cryo-EM) and flexible fitting, we determined the structure of eRF1-eRF3-guanosine 5′- [β,γ-imido] triphosphate (GMPPNP)-bound ribosomal pretermination complex (pre-TC), which corresponds to the initial, pre-GTP hydrolysis stage of factor attachment. Our results show that eukaryotic translation termination involves a network of interactions between the two release factors and the ribosome. Our structure provides mechanistic insight into the coordination between GTP hydrolysis by eRF3 and subsequent peptide release by eRF1.T ermination of translation occurs when a ribosome reaches the end of the coding region and a stop codon (UAA, UAG, or UGA) enters the aminoacyl tRNA binding site (A site), leaving peptidyl-tRNA in the peptidyl tRNA binding site (P site). It entails stop codon recognition by specialized release factors followed by hydrolysis of peptidyl-tRNA. In eukaryotes, termination is mediated by the concerted action of two directly interacting release factors, eRF1 and eRF3. eRF1 is responsible for stop codon recognition and inducing hydrolysis of peptidyl-tRNA, whereas eRF3, a ribosome-dependent GTPase, strongly stimulates peptide release by eRF1 in a GTP-dependent manner (for review, see ref. 1).eRF1 also participates in ribosome recycling: after peptide release it remains associated with the ribosome and together with ATP-binding cassette sub-family E member 1 (ABCE1) promotes splitting the ribosome into free 60S and tRNA/mRNA-associated 40S subunits (2). eRF1 and eRF3 have paralogs, Dom34 (yeast)/ Pelota (mammals) and Hbs1, respectively, which do not participate in termination but instead play a key role in No-go and nonstop decay surveillance mechanisms (e.g., see refs. 3, 4). Dom34/Pelota and Hbs1 do not induce peptide release in a mechanism similar to eRFs. Instead, they cooperate with ABCE1 to promote dissociation of stalled elongation complexes, which is accompanied by peptidyltRNA drop off (5-7). eRF1 comprises N-terminal (N), middle (M), and C-terminal (C) domains (8). The N domain is responsible for stop codon recognition, which is achieved through a 3D network of conserved residues that include apical TASNIKS (amino acid sequence: threonine-alanine-serine-asparagine-isoleucine-lysine-serine) and YxCxxxF motifs (e.g., refs. 9-12). Domain M contains the universally conserved GGQ motif, which is critical for triggering peptide release: as shown for prokaryotes, its placement into the peptidyl transferase center (PTC) causes rRNA rearrangement, allowing a water molecule to enter (for review, see ref. 13). The rigid core of domain C forms an α-β sandwich (8) that deviates from the standard form by the presence of a small insertion, which forms a minidomain (14). eRF3 consists of an N-terminal sequence (residues 1-...

Chemical footprinting reveals conformational changes of 18S and 28S rRNAs at different steps of translation termination on the human ribosome

“…It has been supposed (Hountondji et al 2014) that in all these complexes tRNA could adopt both P/P and P/E configurations and that tRNA in the P/P state is involved in the cross-linking with eRF1, whereas tRNA in the P/E state is cross-linked to the ribosomal protein eL44 (eL36AL). On the contrary, peptidyl-tRNA occupying the P site of the pretermination complex seemed to prevent eRF1 bound to eRF3 and GMPPNP from extending to the PTC and contacting the PTC ring (Taylor et al 2012). Something similar could take place when the pretermination complex was obtained by association of the ternary complex eRF1•eRF3•GMPPNP with the binary 80S…”

“…The GAC including H43 and H44, where nucleotides protected against hydroxyl radicals in eRF3-containing complexes 6 and 7 are located, is not resolved in the cryo-EM models (Muhs et al 2015) as well. However, a contact of this center with eRF1 in the pretermination complex containing eRF1•eRF3•GMPPNP has been observed (Taylor et al 2012). We tried to locate the GAC in the published models Rearrangements in rRNAs at translation termination www.rnajournal.org 281 (Muhs et al 2015) analyzing the cryo-EM structure of the human 80S ribosome (Anger et al 2013), in which the GAC is resolved.…”

“…Analysis of cryo-electron microscopy (cryo-EM) models of mammalian pretermination complexes containing the ternary complex of release factors with a nonhydrolysable GTP analog, eRF1•eRF3•GMPPNP, or eRF1 alone (Taylor et al 2012;des Georges et al 2014;Muhs et al 2015) has led to (1) identification of ribosomal proteins and rRNA heliсes involved in interactions with release factors; (2) discovery of conformational changes induced in eRF1 by GTP hydrolysis-namely, translocation of the universally conserved GGQ loop to the PTC to trigger the peptidyl-tRNA hydrolysis; (3) disclosure of ribosomal structural rearrangements (primarily, inward shift of the P stalk base) caused by binding of eRF1•eRF3 complex, which is believed to be required for GTP hydrolysis; and (4) determination of position of eRF1 bound to the pretermination complex in the absence of eRF3. However, cryo-EM structures provided no data on particular rRNA nucleotides involved in interactions with eRF1 and eRF3 in mammalian pretermination complexes.…”

Taura syndrome virus IRES initiates translation by binding its tRNA-mRNA–like structural element in the ribosomal decoding center

“…When eRF1 was added together with the translocase eEF-2, translocation by one codon was observed (17). Because release factors bind to the ribosomal A site in response to a stop codon located in the decoding center (51,52), these experiments demonstrate that PKI is originally positioned in the A site. Upon translocation, the following UAA codon is brought into the A site, allowing codon-specific binding of eRF1.…”

Functional analysis of the uL11 protein impact on translational machinery

“…In eukaryotic cells, recognition of the STOP codon is subjected to the decoding process analogously to TC recognition, with the eRF1:eRF3:GTP complex (termination ternary complex tTC) as a main player, which structurally mimics TC, where eRF3 works as trGTPase analogously to EF-Tu. 56 Similarly to the decoding event during elongation, eRF1 binding to the STOP codon is disfavored when uL11 is absent, and near-cognate TC binding may take place, making termination less accurate. Thus, uL11-depleted cells lose this discriminative capacity, equalizing the probability for cognate, near-cognate, and termination TC binding.…”