Strukturen von eEF3 und Sec61 als aktive ribosomale Liganden
This work presents cryo-EM structures of two active ribosomal ligands in yeast, namely eEF3 and Sec61. Protein synthesis requires two canonical elongation factors in all kingdoms. These are the GTPases eEF1A (EF-Tu in prokaryotes) and eEF2 (EF-G in prokayotes). In fungi, a third elongation factor eEF3 is required, which belongs to the ATP-binding cassette- (ABC-) protein family. This factor is essential for the binding of the aminoacyl-tRNA-eEF1A-GTP ternary complex to the A site of the ribosome and has been suggested to do so by facilitating the clearance of deacyl-tRNA from the ribosomal E-site. The cryo-EM structure of the ATP-bound form of eEF3 in complex with an 80S ribosome shows that eEF3 binds the ribosome in the post-translocational conformation using a novel binding site. From there the affinity for tRNA in the ribosomal E site could be easily modified. Using the crystal structure of eEF3, a molecular model for eEF3 was generated, which provides the structural basis for the activity of an ABC protein in the context of translation. In co-translational translocation the trimeric Sec61-complex serves as a signal sequence-gated protein-conducting channel (PCC) which allows the translocation of a secetory protein through and the integration of a membrane protein into the lipid bilayer of the ER. Bound to a ribosome, the active PCC seems to have an oligomeric structure consisting of several copies of Sec61-trimers. The crystal structure of an inactive PCC, however, shows only a single heterotrimer (monomer) which could also be functional in the active state. The cryo-EM structure of the active PCC in complex with a translating ribosome shows the hitherto most detailed electron density map for the PCC. The most characteristic feature is a funnel-shaped off-centered pore at the cytosolic side. Several models for the active state were docked into the EM-density. Although a precise molecular interpretation is limited by the resolution (12, 3 Å), a single copy of an opened Sec61-heterotrimer seems to be the most reasonable fit for the density.
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School:Humboldt-Universität zu Berlin
Source Type:Master's Thesis
Date of Publication:01/30/2007