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Schaffitzel Team

Ribosomal complexes: targeting, translocation and quality control

Schaffitzel Team

Figure 1: EM reconstruction of the ribosome nascent chain complex and the translocon. The 30S ribosomal subunit is shown in yellow, the 50S subunit in light blue, the A, P and E site tRNAs are in magenta, green and orange respectively. The mRNA is depicted in cyan and the nascent chain in the polypeptide exit tunnel in green. The active translocon in dark blue is located at the exit of the ribosomal tunnel. A second, non-translocating translocon (red) is bound to the mRNA

Schaffitzel Team

Figure 2: Cryo-EM structure of the ribosome (yellow/blue) bound to the signal recognition particle (SRP) and the SRP receptor (both in red). Below it is an atomic model of SRP (green-yellow/ orange) and its receptor (pink)

The Schaffitzel team combines molecular biology, biochemistry and cryo-electron microscopy to study large macromolecular complexes in protein targeting, secretion and membrane protein integration.

Previous and current research

Research in our laboratory combines molecular biology, in vitro and in vivo biochemistry, and single-particle cryo-electron microscopy (cryo-EM) to study the structure and function of ribosomal complexes. Synthesis and folding of proteins require concerted interactions of the translating ribosome with translation factors, regulatory factors, molecular chaperones, and factors involved in the export of proteins. Structures of translating ribosomes in complex with these factors provide critical insight into the interaction networks, stoichiometry, and molecular mechanisms of these megadalton-size complexes. Using cryo-EM, we can study the multi-component translation machinery at close to physiological conditions. By using state-of-the-art electron microscopes and by image processing of large data sets, EM structures of prokaryotic and eukaryotic ribosomes have been obtained at subnanomolar resolution, demonstrating the power of this method.

A prerequisite for our functional and structural studies is the production of large amounts of homogenous, stable complexes in the quantity and quality required for interaction assays, mass spectrometry and single-particle cryo-EM. We have established bacterial and eukaryotic cell-free translation systems for the in vitro generation of ribosomes that display homogenous nascent polypeptide chains or have stalled at a defined step in translation. We reconstitute the ribosomal complexes along the pathways of co-translational targeting and translocation, and mRNA quality control. This approach was successfully applied in the case of the cryo-EM structures of the complex of the ribosome with the translocation machinery SecYEG (figure 1), of the translating ribosome-signal recognition particle (SRP) complex, and of the ribosome in complex with SRP and SRP receptor (figure 2). The data from intermediate resolution structures derived from cryo-EM, in conjunction with high-resolution structures of the ribosome and of the isolated factors, were combined in a hybrid approach to generate quasi-atomic models of the ribosomal complexes involved. The structural data, supported by biochemical data, provide important and detailed snapshots of the mechanisms underlying these cellular processes, ensuring correct folding, targeting and translocation of nascent proteins.

Future projects and goals

We study ribosomal complexes involved in targeting, membrane protein integration, folding, and assembly. We analyse the membrane protein complexes biochemically, by cross-linking/mass spectrometry (collaboration with Juri Rappsilber, TU Berlin) and single-particle cryo-electron microscopy. In collaboration with the Cusack, Hentze and Kulozik (MMPU) groups, we study mammalian ribosomal complexes involved in nonsense-mediated mRNA decay. We produce the eukaryotic factors involved using advanced recombinant eukaryotic technologies in collaboration with the Berger group. Finally, our team collaborates with a number of groups to solve the structures of large macromolecular complexes in transcription, epigenetics and cellular signalling.