Strukturelle Basis kanonischer und nicht-kanonischer Translationstermination und Ribosomenrecycling durch eRF1/eRF3 und ABCE1
Zusammenfassung der Projektergebnisse
The major goal of the proposal was to understand eukaryotic translation termination and ribosome recycling on a molecular level. Our strategy was to assemble pre-termination, termination/prerecycling and post-recycling complexes from baker’s yeast, Neurospora, wheat germ and human in vitro systems and determine cryo-EM structures at the best possible resolution. To position a stop codon in the ribosomal A site, we programmed ribosomes with a mRNA derived from the cytomegalovirus (CMV) gp48 uORF2 in in vitro translation extracts. uORF2 encodes a the translation termination stalling sequence and its translation results with a stop codon in the A site. In the first funding period, we obtained first structural insights revealing the overall architecture of CMV-stalled 80S-eRF1-eRF3 and 80S-eRF1-ABCE1 complexes. We could describe the conformational dynamics of eRF1 when transitioning from a pre-termination to the termination/prerecycling state. Stabilization of the eRF1 extended conformation by ABCE1 could explain the tremendously faster kinetics of the termination reaction in presence of the recycling factor. The highly conserved GGQ motif – the only common sequence with bacterial release factors – is positioned ideally beneath the CCA-end of the peptidyl-tRNA to catalyse the hydrolysis of the nascent peptide from the P site tRNA. Moreover, we could show that canonical termination and ribosome rescue after aberrant elongation follow a similar mechanochemical mechanism of ABCE1-dependent ribosome recycling. A high resolution structure of the human CMV-RNC-eRF1 complex revealed, how eRF1 recognizes stop codons. It revealed that the stop codon forms a unique U-turn involving the fourth base, thus forming a base quadruplet. The quadruplet is stabilized by conserved bases in the ribosomal A site and is recognized by a specific binding pocket formed by the eRF1 N-terminal domain. In addition, this structure explained the mechanism of peptide release silencing caused by the CMV stalling peptide. Whereas the ester bond between A76 and the terminal residue as well as the eRF1 the GGQ-loop are in a canonical release-competent position, the peptidyl-transferase center is disturbed by the CMV stalling peptide, which form an α-helix in the upper part of the exit tunnel. Beyond eRF1-dependent termination we further investigated the role of ABCE1 in dissociating the 80S into 40S and 60S subunits. We determined a high resolution cryo-EM structure of a 40S-ABCE1 postrecycling complex that revealed conformational changes in ABCE1 corroborating and refining our model for ribosome recycling. While the nucleotide binding sites (NBS) are in a closed, ATP-occluded conformation, the FeS domain relocates towards the ribosomal A site. In its new position it acts as an anti-association factor for the 60S subunit. Moreover the direction of the FeS domain movement confirms the model in which the energy of NBS closure and/or ATP-hydrolysis is transmitted via the FeS domain to eRF1, which acts as a wedge to split the 80S. Additionally our work suggests a role of 30S/40S-bound ABCE1 during translation initiation or re-initiation. Beyond the termination project, numerous collaborations within the FOR1805 environment were started, mainly on the aspect of translation stalling (with the Wilson, Rodnina, Grübmüller/Vaiana groups) but also on ribosome rescue and antibiotic resistance (with the Wilson group) and ribosome hibernation (with the Wilson and Ignatova groups).
Projektbezogene Publikationen (Auswahl)
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Cryoelectron microscopic structures of eukaryotic translation termination complexes containing eRF1-eRF3 or eRF1-ABCE1. Cell Rep. 8, 59-65 (2014)
Preis, A., Heuer, A., Barrio-Garcia, C., Hauser, A., Eyler, D.E., Berninghausen, O., Green, R., Becker, T., Beckmann, R.
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Structure of a human translation termination complex. Nucleic Acids Res. 43, 8615-26, (2017)
Matheisl, S., Berninghausen, O., Becker, T., Beckmann, R.
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A combined cryo-EM and molecular dynamics approach reveals the mechanism of ErmBL-mediated translation arrest. Nat. Commun. 27:12026 (2016)
Arenz, S., Bock, L.V., Graf, M., Innis, C.A., Beckmann, R., Grubmüller, H., Vaiana, A.C., Wilson, D.N.
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Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome. Nucleic Acids Res. 44, 1944-51, (2016)
Schmidt, C., Becker, T., Heuer, A., Braunger, K., Shanmuganathan, V., Pech, M., Berninghausen, O., Wilson, D.N., Beckmann R.
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The cryo-EM structure of a ribosome-Ski2-Ski3-Ski8 helicase complex. Science. 354, 1431-1433, (2016)
Schmidt, C., Kowalinski, E., Shanmuganathan, V., Defenouillère, Q., Braunger, K., Heuer, A., Pech, M., Namane, A., Berninghausen, O., Fromont-Racine, M., Jacquier, A., Conti, E., Becker, T., Beckmann, R.
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Translation regulation via nascent polypeptidemediated ribosome stalling. Curr Opin Struct Biol. 37:123-33, (2016)
Wilson, D.N., Arenz, S., Beckmann, R.
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Cryo-EM structure of a late pre-40S ribosomal subunit from Saccharomyces cerevisiae. Elife. pii: e30189 (2017)
Heuer, A., Thomson, E., Schmidt, C., Berninghausen, O., Becker, T., Hurt, E., Beckmann, R.
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Ribosome Structural Basis for Polyproline-Mediated Stalling and Rescue by the Translation Elongation Factor EF-P. Mol Cell. 68, 515-527 (2017)
Huter, P., Arenz, S., Bock, L.V., Graf, M., Frister, J.O., Heuer, A., Peil, L., Starosta, A.L., Wohlgemuth, I., Peske, F., Nováček, J., Berninghausen, O., Grubmüller, H., Tenson, T., Beckmann, R., Rodnina, M.V., Vaiana, A.C., Wilson, D.N.
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Structure of the 40S-ABCE1 post-splitting complex in ribosome recycling and translation initiation. Nat Struct Mol Biol. 24, 453-460, (2017)
Heuer, A., Gerovac, M., Schmidt, C., Trowitzsch, S., Preis, A., Kötter, P., Berninghausen, O., Becker, T., Beckmann, R., Tampé, R.
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The force-sensing peptide VemP employs extreme compaction and secondary structure formation to induce ribosomal stalling. Elife. pii: e25642, (2017)
Su,T., Cheng, J., Sohmen, D., Hedman, R., Berninghausen, O., von Heijne, G., Wilson, D.N., Beckmann, R.
