Structural and Functional Insights into Dom34, a Key Component of No-Go mRNA Decay

103

172

No-go decay and nonstop decay are mRNA surveillance pathways that detect translational stalling and degrade the underlying mRNA, allowing the correct translation of the genetic code. In eukaryotes, the protein complex of Pelota (yeast Dom34) and Hbs1 translational GTPase recognizes the stalled ribosome containing the defective mRNA. Recently, we found that archaeal Pelota (aPelota) associates with archaeal elongation factor 1α (aEF1α) to act in the mRNA surveillance pathway, which accounts for the lack of an Hbs1 ortholog in archaea. Here we present the complex structure of aPelota and GTP-bound aEF1α determined at 2.3-Å resolution. The structure reveals how GTP-bound aEF1α recognizes aPelota and how aPelota in turn stabilizes the GTP form of aEF1α. Combined with the functional analysis in yeast, the present results provide structural insights into the molecular interaction between eukaryotic Pelota and Hbs1. Strikingly, the aPelota·aEF1α complex structurally resembles the tRNA·EF-Tu complex bound to the ribosome. Our findings suggest that the molecular mimicry of tRNA in the distorted "A/T state" conformation by Pelota enables the complex to efficiently detect and enter the empty A site of the stalled ribosome.X-ray crystallography | small G protein | dual specificity

Section: Resultsmentioning

confidence: 62%

mentioning

confidence: 99%

Structural basis for mRNA surveillance by archaeal Pelota and GTP-bound EF1α complex

Kobayashi¹,

Kikuno²,

Kuroha

et al. 2010

103

172

“…Although in contrast to the Dom34-Hbs1-GTP mRNA surveillance complex, which targets stalled elongation complexes in an A-site codon-independent manner (5, 7), the eRF1-eRF3-GTP complex recognizes stop codons exclusively, and the N domains of eRF1 and Dom34 are not structurally related (8,40), in both cases the N domains of eRF1 and Dom34 penetrate deeply into the decoding center and establish multiple interactions with the 40S subunit (present study and ref. 29).…”

Section: Discussionmentioning

confidence: 99%

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

Taylor

Unbehaun

et al. 2012

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-...

“…Dom34 and Hbs1, originally identified in the No-Go Decay pathway (27), share significant structural similarity with the canonical eukaryotic release factors (28)(29)(30)(31), but lack the residues necessary for both stop codon recognition and hydrolysis of peptidyl-tRNA (32). Like Rli1, eukaryotic release (eRF1, eRF3) and release-like (Dom34, Hbs1) factors are highly conserved from archaea to metazoans, albeit with the exception that the translational GTPases eRF3 and Hbs1 appear to be functionally replaced by the related GTPase aEF1α in archaea (33,34).…”

mentioning

confidence: 99%

Kinetic analysis reveals the ordered coupling of translation termination and ribosome recycling in yeast

Shoemaker

Green

2011

235

318

Although well defined in bacterial systems, the molecular mechanisms underlying ribosome recycling in eukaryotic cells have only begun to be explored. Recent studies have proposed a direct role for eukaryotic termination factors eRF1 and eRF3 (and the related factors Dom34 and Hbs1) in downstream recycling processes; however, our understanding of the connection between termination and recycling in eukaryotes is limited. Here, using an in vitro reconstituted yeast translation system, we identify a key role for the multifunctional ABC-family protein Rli1 in stimulating both eRF1-mediated termination and ribosome recycling in yeast. Through subsequent kinetic analysis, we uncover a network of regulatory features that provides mechanistic insight into how the events of termination and recycling are obligately ordered. These results establish a model in which eukaryotic termination and recycling are not clearly demarcated events, as they are in bacteria, but coupled stages of the same release-factor mediated process.peptide release | ABCE1 | Pelota T he translation of messenger RNA into protein is traditionally thought to consist of four discrete steps: initiation, elongation, termination, and recycling (1, 2). Recycling, the final stage of translation, involves subunit dissociation and recovery of translational components (e.g., mRNA, tRNA, etc.) for reuse in subsequent rounds of translation. In bacteria, recycling depends on a specialized ribosome recycling factor (RRF) and elongation factor G, which together separate subunits in a GTP-dependent manner (3, 4). These factors selectively act on posttermination complexes following the facilitated removal of class 1 release factors (RF1 or RF2) by the class 2 release factor GTPase (RF3) (5). The departure of the termination factors ensures that ribosome recycling does not commence until after completion of peptide release. Separated subunits are subsequently bound by various initiation factors, including IF3, which prevent reassociation of subunits, allowing for the dissociation of mRNA and tRNA species and preparing subunits for the next round of initiation (3).Neither RRF nor RF3 are conserved outside of bacteria, suggesting that this mode of recycling is also not conserved. Rather, a highly conserved ABC-family ATPase, ABCE1, was recently implicated in ribosome recycling in both eukaryotic and archaeal systems (6, 7). ABCE1 (or Rli1 in yeast) is a cytosolic ABC-family ATPase containing two NTP binding domains and a conserved Nterminal [4Fe-4S] domain that is required for its function (7-10). Characteristic of this family of ATPases, Rli1 is thought to convert the chemical energy of ATP hydrolysis into a mechanical tweezer-like motion (11). It is highly conserved throughout eukaryotes and archaea (12, 13) and is essential in all organisms tested (14-16). Consistent with this, Rli1 has been implicated in several essential, conserved cellular processes including ribosome maturation, translation initiation, and translation termination, with additional roles in RNAse L i...