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Structural basis of translational recycling and bacterial ribosome rescue
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.
Ribosome, cryo-EM, Ribosome Recycling, ABCE1, Ribosome Rescue
Kratzat, Hanna
2022
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Kratzat, Hanna (2022): Structural basis of translational recycling and bacterial ribosome rescue. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

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.