Structural basis of canonical and non-canonical translation termination and recycling by eRF1/eRF3 and ABCE1 in yeast and humans

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Project P3 (Beckmann) addresses the essential phases of eukaryotic translation termination and ribosome recycling using the yeast (S. cerevisiae) and the human system. In the first funding period functional termination complexes (containing eRF1 and eRF3) and pre-recycling complexes (containing eRF1 and ABCE1) were generated using a wheat germ in vitro translation system. To position a stop-codon in the ribosomal A-site a viral polypeptide with stalling sequence was used. Such stalled ribosomes were in vitro reconstituted with purified termination/recycling factors and cryo-EM structures of assembled complexes were solved at 8-9 Å resolution. We observe that after decoding the stop-codon in the eRF3-containing pre-termination complex the eRF1 central domain moves into the peptidyl-transferase center of the large 60S subunit to release the nascent peptide in the ABCE1-containing complex. This explains how eRF1 functions in both translation termination and ribosome recycling on a structural level. To obtain similar structures in the human system we established a reliable and highly efficient human in vitro translation system. Furthermore we optimized the expression for constructs yielding stalled ribosomes by using the initiation factor independent CrPV-IRES. On this basis termination and pre-recycling complexes could be reconstituted in vitro resulting in first promising cryo-EM reconstructions. In the second funding period we will continue to elucidate the molecular basis of human termination and recycling by high-resolution cryo-EM (using newly installed FEI Falcon 2 direct electron detector technology) with a focus on stop-codon recognition and peptidyl-tRNA hydrolysis by eRF1.