Structural basis of translational recycling and bacterial ribosome rescue

In the last step of gene expression, a messenger RNA (mRNA) sequence is translated into a polypeptide. This highly regulated and dynamic process is carried out by the ribosome, a ribonucleoprotein complex composed of two unequal subunits. The translation cycle is initiated when the small ribosomal subunit (SSU) binds to an mRNA and recognizes the start codon of the open reading frame (ORF). Then the large ribosomal subunit (LSU) joins and the ribosome starts moving along the mRNA. A protein is synthesized until the ribosome reaches a stop codon. A cell needs thousands (prokaryotes) or millions (eukaryotes) of ribosomes for protein production and spends enormous amounts of energy on the assembly of this macromolecular machinery. Therefore, it is crucial to recycle the machinery after each successful round of translation. The recycling step allows release of mRNA, transfer RNA (tRNA) and the synthesized polypeptide from ribosomal subunits and subsequent binding of the next mRNA for protein synthesis. The first part of this dissertation includes studies of the highly conserved and essential ribosome recycling factor ATP binding cassette (ABC) Subfamily E Member 1 (ABCE1). In eukaryotes and archaea, ABCE1 binds the ribosome and in concert with an A-site factor and splits the ribosome into large and small subunits. ABCE1 harbors two nucleotide binding sites (NBSs), which are formed at the interface of two nucleotide binding domains (NBDs). Prior to this work, the ABCE1-bound pre-splitting complex, as well as the ABCE1-bound post-splitting complex, had been visualized by cryo-electron microscopy (cryo-EM) at medium resolution. This structural analysis combined with functional studies led to a model for the mechanism of the splitting event. ATP-binding and the closure of the NBSs lead to repositioning of the iron-sulfur cluster domain, which results in collision with the A-site factor and ribosome splitting. Yet, how conformational changes during the splitting event are triggered and communicated to the NBSs of ABCE1, was not understood. To gain molecular insights into this process, a structure of a fully nucleotide-occluded (closed) state of ABCE1 bound to the archaeal 30S post-splitting complex was solved by cryo-EM. At a resolution of 2.8 Å a detailed molecular analysis of ABCE1 was performed and confirmed by a combination of mutational and functional studies. This allowed to propose a refined model of how the ATPase cycle is linked to ribosome splitting and which role the different domains of ABCE1 play. In eukaryotes, the recycling phase is directly linked to translation initiation via the SSU. After being released from the mRNA 3’ end, the SSU can engage with another or even the same mRNA at the 5’ end. The recycling factor ABCE1 was found to be associated with initiation complexes, but whether it plays a role in initiation was not clear. Using cryo-EM, structures of native ABCE1-containing initiation complexes were solved and intensive 3D classification allowed to distinguish different stages of initiation, during which ABCE1 may play a role. Surprisingly, ABCE1 adopted a previously unknown state for ABC-type ATPases that was termed “hybrid state”. Here, the NBSI is in a half open state with ADP bound and the NBSII is in a closed state with ATP bound. Further, eukaryotic initiation factor 3j (eIF3j) was found to stabilize this hybrid conformation via its N-terminus. Since eIF3j had already been described to assist ABCE1 in ribosome dissociation, in vitro splitting assays were performed demonstrating that eiF3j indeed actively enhances the splitting reaction. On top of this, the high-resolution structure allowed to describe the interaction network of eIF3j with the ribosome, initiation factors (IFs), and ABCE1. Independent of ABCE1, the structures presented here allowed to provide an improved molecular model of the human 43S pre-initiation complex (PIC) and to analyze its sophisticated interaction network. In particular, new molecular insights into the large eIF3 complex encircling the 43S PIC, and the eIF2 ternary complex delivering the initiator tRNA are provided. Equally important as canonical recycling is the recognition and recycling of ribosomes that result from translational failure. Aberrant translation elongation and ribosome stalling can be caused by a plethora of different stresses. In bacterial cells, multiple rescue systems are known such as trans-translation or alternative ribosome rescue factor-mediated termination, which act on ribosome nascent chain complexes with an empty A-site (non-stop complexes). It has been a long standing question how ribosomes that are stalled in the middle of an ORF (no-go complexes) are recognized and recycled. The second part of this dissertation reports a new bacterial rescue system that acts on no-go complexes. In eukaryotes, the concept of ribosome collisions as a trigger for ribosome rescue has been studied extensively. Here, it was found that a similar mechanism exists in bacteria and thus a structural analysis of collided disomes in E. coli and B. subtilis was conducted. In a genetic screen, the endonuclease SmrB was identified as one candidate for a collision sensor. Structural analysis of SmrB-bound disomes elucidated how this rescue factor is recruited to collided ribosomes. Its SMR domain binds to the disome interface between the stalled and the collided ribosome in close proximity to the mRNA and in a position ideal to perform endonucleolytic cleavage. Such cleavage then results in non-stop complexes that can be recycled by the pathways mentioned above. In conclusion, this work provides mechanistic insights into how a cell distinguishes stalled ribosomes from actively translating ribosomes and characterizes a novel ribosome rescue pathway

Visualization of chemical modifications in the human 80S ribosome structure

Comment in RNA modifications: Ribosomes get decorated. [Nat Chem Biol. 2017]International audienceChemical modifications of human ribosomal RNA (rRNA) are introduced during biogenesis and have been implicated in the dysregulation of protein synthesis, as is found in cancer and other diseases. However, their role in this phenomenon is unknown. Here we visualize more than 130 individual rRNA modifications in the three-dimensional structure of the human ribosome, explaining their structural and functional roles. In addition to a small number of universally conserved sites, we identify many eukaryote- or human-specific modifications and unique sites that form an extended shell in comparison to bacterial ribosomes, and which stabilize the RNA. Several of the modifications are associated with the binding sites of three ribosome-targeting antibiotics, or are associated with degenerate states in cancer, such as keto alkylations on nucleotide bases reminiscent of specialized ribosomes. This high-resolution structure of the human 80S ribosome paves the way towards understanding the role of epigenetic rRNA modifications in human diseases and suggests new possibilities for designing selective inhibitors and therapeutic drugs

Atomic model building and refinement into high-resolution cryo-EM maps

International audienc

Visualization of chemical modifications in the human 80S ribosome structure

Video showing the 3D structure of the human ribosome revealing chemical modifications of the ribosomal RNA.<br><div><br></div><div>File is provided in .mp4 format, compatible with most standard video playing software.</div><div><br></div><div>This visualization, along with the related article, presents for the first time chemical modifications of rRNA in the human ribosome introduced during biogenesis and maturation, and highlights their structural and functional role within their 3D molecular environment. Visualizations include:</div><div><br></div><div>Universally conserved rRNA modification sites (Class I)</div><div>Predicted rRNA modification sites (Class II)</div><div>Unpredicted human specific rRNA modifications (Class III)</div><div><br></div><div><div>The structure of the human 80S ribosome was determined by single particle cryo electron microscopy (cryo-EM) and refined using focused refinement of the 60S ribosomal subunit and the 40S head and body parts during image processing (resolved to 2.9, 3.0 and 3.1 Å average resolution, respectively (see methods in linked article).</div></div><div><br></div><div>See background below and related article linked from this data record for further details.</div><div><br></div><div><b>Background:</b></div><div>Chemical modifications of the ribosomal RNA (rRNA) of the human ribosome are introduced during biogenesis and are implicated in human protein synthesis dysregulations such as cancer and other diseases but their role therein is unknown. Here we visualize over 130 individual rRNA modifications in the three dimensional structure of the human ribosome explaining their structural and functional roles. Beyond some universally conserved sites, many eukaryote/human specific modifications and new unique sites are found that form an evolutionary extended shell compared to bacterial ribosomes and which stabilize the RNA. A series of modifications are located in vicinity to 3 bound antibiotics or are associated with degenerated states in cancer such as keto alkylations at nucleotide bases reminiscent of specialized ribosomes. This high-resolution structure of the human 80S ribosome paves the way to understanding the role of rRNA modifications in human diseases and drug-design. <br></div

Focused classification and refinement in high-resolution cryo-EM structural analysis of ribosome complexes

International audienceCryo electron microscopy (cryo-EM) historically has had a strong impact on the structural and mechanistic analysis of protein synthesis by the prokaryotic and eukaryotic ribosomes. Vice versa, studying ribosomes has helped moving forwards many methodological aspects in single particle cryo-EM, at the level of automated data collection and image processing including advanced techniques for particle sorting to address structural and compositional heterogeneity. Here we review some of the latest ribosome structures, where cryo-EM allowed gaining unprecedented insights based on 3D structure sorting with focused classification and refinement methods helping to reach local resolution levels better than 3Å. Such high-resolution features now enable the analysis of drug interactions with RNA and protein side-chains including even the visualization of chemical modifications of the ribosomal RNA. These advances represent a major breakthrough in structural biology and show the strong potential of cryo-EM beyond the ribosome field including for structure-based drug design

Molecular analysis of the ribosome recycling factor ABCE1 bound to the 30S post-splitting complex

Ribosome recycling by the twin-ATPase ABCE1 is a key regulatory process in mRNA translation and surveillance and in ribosome-associated protein quality control in Eukarya and Archaea. Here, we captured the archaeal 30S ribosome post-splitting complex at 2.8 Å resolution by cryo-electron microscopy. The structure reveals the dynamic behavior of structural motifs unique to ABCE1, which ultimately leads to ribosome splitting. More specifically, we provide molecular details on how conformational rearrangements of the iron–sulfur cluster domain and hinge regions of ABCE1 are linked to closure of its nucleotide-binding sites. The combination of mutational and functional analyses uncovers an intricate allosteric network between the ribosome, regulatory domains of ABCE1, and its two structurally and functionally asymmetric ATP-binding sites. Based on these data, we propose a refined model of how signals from the ribosome are integrated into the ATPase cycle of ABCE1 to orchestrate ribosome recycling

Structure and function of yeast Lso2 and human CCDC124 bound to hibernating ribosomes.

Cells adjust to nutrient deprivation by reversible translational shutdown. This is accompanied by maintaining inactive ribosomes in a hibernation state, in which they are bound by proteins with inhibitory and protective functions. In eukaryotes, such a function was attributed to suppressor of target of Myb protein 1 (Stm1; SERPINE1 mRNA-binding protein 1 [SERBP1] in mammals), and recently, late-annotated short open reading frame 2 (Lso2; coiled-coil domain containing short open reading frame 124 [CCDC124] in mammals) was found to be involved in translational recovery after starvation from stationary phase. Here, we present cryo-electron microscopy (cryo-EM) structures of translationally inactive yeast and human ribosomes. We found Lso2/CCDC124 accumulating on idle ribosomes in the nonrotated state, in contrast to Stm1/SERBP1-bound ribosomes, which display a rotated state. Lso2/CCDC124 bridges the decoding sites of the small with the GTPase activating center (GAC) of the large subunit. This position allows accommodation of the duplication of multilocus region 34 protein (Dom34)-dependent ribosome recycling system, which splits Lso2-containing, but not Stm1-containing, ribosomes. We propose a model in which Lso2 facilitates rapid translation reactivation by stabilizing the recycling-competent state of inactive ribosomes

A structural inventory of native ribosomal ABCE1‐43S pre‐initiation complexes

International audienceIn eukaryotic translation, termination and ribosome recycling phases are linked to subsequent initiation of a new round of translation by persistence of several factors at ribosomal sub-complexes. These comprise/include the large eIF3 complex, eIF3j (Hcr1 in yeast) and the ATP-binding cassette protein ABCE1 (Rli1 in yeast). The ATPase is mainly active as a recycling factor, but it can remain bound to the dissociated 40S subunit until formation of the next 43S pre-initiation complexes. However, its functional role and native architectural context remains largely enigmatic. Here, we present an architectural inventory of native yeast and human ABCE1-containing pre-initiation complexes by cryo-EM. We found that ABCE1 was mostly associated with early 43S, but also with later 48S phases of initiation. It adopted a novel hybrid conformation of its nucleotide-binding domains, while interacting with the N-terminus of eIF3j. Further, eIF3j occupied the mRNA entry channel via its ultimate C-terminus providing a structural explanation for its antagonistic role with respect to mRNA binding. Overall, the native human samples provide a near-complete molecular picture of the architecture and sophisticated interaction network of the 43S-bound eIF3 complex and the eIF2 ternary complex containing the initiator tRNA

An ATP-dependent partner switch links flagellar C-ring assembly with gene expression

Bacterial flagella differ in their number and spatial arrangement. In many species, the MinD-type ATPase FlhG (also YlxH/FleN) is central to the numerical control of bacterial flagella, and its deletion in polarly flagellated bacteria typically leads to hyperflagellation. The molecular mechanism underlying this numerical control, however, remains enigmatic. Using the model species Shewanella putrefaciens, we show that FlhG links assembly of the flagellar C ring with the action of the master transcriptional regulator FlrA (named FleQ in other species). While FlrA and the flagellar C-ring protein FliM have an overlapping binding site on FlhG, their binding depends on the ATP-dependent dimerization state of FlhG. FliM interacts with FlhG independent of nucleotide binding, while FlrA exclusively interacts with the ATP- dependent FlhG dimer and stimulates FlhG ATPase activity. Our in vivo analysis of FlhG partner switching between FliM and FlrA reveals its mechanism in the numerical restriction of flagella, in which the transcriptional activity of FlrA is down-regulated through a negative feedback loop. Our study demonstrates another level of regulatory complexity underlying the spationumerical regulation of flagellar biogenesis and implies that flagellar assembly transcriptionally regulates the production of more initial building blocks