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Cryo-EM structures of eukaryotic translation termination and ribosome recycling complexes containing eRF1, eRF3 and ABCE1
Cryo-EM structures of eukaryotic translation termination and ribosome recycling complexes containing eRF1, eRF3 and ABCE1
Translation of an mRNA template into a polypeptide chain is terminated on a stop codon. The stop is recognized in the ribosomal A site by release factors, that subsequently release the polypeptide. This event is followed by ribosome recycling leading to a dissociation of the ribosome into subunits. Eukaryotic eRF1 recognizes all three stop codons and is delivered to the ribosomal A site in a ternary complex with GTP-bound eRF3. ABCE1, a highly conserved ATPase, stimulates peptide release and splits the ribosome in concert with eRF1. The first goal of this work was to study the structural rearrangements of eRF1, eRF3 and ABCE1 on the ribosome during translation termination and ribosome recycling. Two cryo-EM structures were obtained at sub-nanometer resolution: the pre-termination complex containing eRF1 and eRF3, and a termination/pre-recycling complex containing eRF1 and ABCE1. The pre-termination stage showed eRF1 packed against eRF3, unable to catalyze peptide release. In the termination/pre-recycling complex, eRF1 assumed an extended conformation which is further stabilized by ABCE1, with the central domain of eRF1 swung out toward the CCA end of the P-site tRNA. ABCE1 adopted a half-closed conformation of its two nucleotide-binding domains in the termination/pre-recycling complex. According to a model based of these results, splitting the ribosome would require the closing of the two nucleotide-binding domains and a rotation of the iron-sulfur cluster domain of ABCE1, which would in turn push eRF1 into the intersubunit space. Supporting this idea, ABCE1 was shown to remain bound to the small ribosomal subunit after in vitro splitting. 40S-bound ABCE1 adopted a fully closed conformation and in which re-association of the large ribosomal subunit is prevented. As a second goal of this work, native S. cerevisiae ABCE1-bound small ribosomal subunits were purified to complement the in vitro studies and explore the supposed involvement of ABCE1 in translation initiation. Cryo-EM of native 40S-ABCE1 complexes indeed confirmed the closed conformation of ABCE1. Moreover, these complexes were associated with initiator tRNA and eIF1A, an initiation factor which binds the ribosomal A site and is involved in multiple processes in initiation including subunit joining. These results are clearly hinting at an active role of ABCE1 during translation initiation. Yet, the exact role of ABCE1 will be subject of further studies.
Translation, translation termination, ribosome recycling
Preis, Anne
2020
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Preis, Anne (2020): Cryo-EM structures of eukaryotic translation termination and ribosome recycling complexes containing eRF1, eRF3 and ABCE1. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Translation of an mRNA template into a polypeptide chain is terminated on a stop codon. The stop is recognized in the ribosomal A site by release factors, that subsequently release the polypeptide. This event is followed by ribosome recycling leading to a dissociation of the ribosome into subunits. Eukaryotic eRF1 recognizes all three stop codons and is delivered to the ribosomal A site in a ternary complex with GTP-bound eRF3. ABCE1, a highly conserved ATPase, stimulates peptide release and splits the ribosome in concert with eRF1. The first goal of this work was to study the structural rearrangements of eRF1, eRF3 and ABCE1 on the ribosome during translation termination and ribosome recycling. Two cryo-EM structures were obtained at sub-nanometer resolution: the pre-termination complex containing eRF1 and eRF3, and a termination/pre-recycling complex containing eRF1 and ABCE1. The pre-termination stage showed eRF1 packed against eRF3, unable to catalyze peptide release. In the termination/pre-recycling complex, eRF1 assumed an extended conformation which is further stabilized by ABCE1, with the central domain of eRF1 swung out toward the CCA end of the P-site tRNA. ABCE1 adopted a half-closed conformation of its two nucleotide-binding domains in the termination/pre-recycling complex. According to a model based of these results, splitting the ribosome would require the closing of the two nucleotide-binding domains and a rotation of the iron-sulfur cluster domain of ABCE1, which would in turn push eRF1 into the intersubunit space. Supporting this idea, ABCE1 was shown to remain bound to the small ribosomal subunit after in vitro splitting. 40S-bound ABCE1 adopted a fully closed conformation and in which re-association of the large ribosomal subunit is prevented. As a second goal of this work, native S. cerevisiae ABCE1-bound small ribosomal subunits were purified to complement the in vitro studies and explore the supposed involvement of ABCE1 in translation initiation. Cryo-EM of native 40S-ABCE1 complexes indeed confirmed the closed conformation of ABCE1. Moreover, these complexes were associated with initiator tRNA and eIF1A, an initiation factor which binds the ribosomal A site and is involved in multiple processes in initiation including subunit joining. These results are clearly hinting at an active role of ABCE1 during translation initiation. Yet, the exact role of ABCE1 will be subject of further studies.