Schaffitzel Team
Ribosomal complexes: targeting, translocation and quality control
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.
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).
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 mechanism of these megadalton-size complexes. With 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 singleparticle cryo-EM. In our laboratory, we established bacterial and eukaryotic cell-free translation systems for the in vitro generation of ribosomes displaying homogenous nascent polypeptide chains or stalled at a defi ned 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 nanoelectrospray mass spectrometry (collaboration with Carol Robinson, University of Oxford and cryo-electron microscopy.
In collaboration with the groups of Stephen Cusack, Matthias Hentze and Andreas Kulozik (MMPU), we study mammalian ribosomal complexes involved in nonsense-mediated mRNA decay. We produce the eukaryotic factors involved by means of advanced recombinant eukaryotic technologies in collaboration with the Berger group.
ERC Investigator
Podcast on Cryo-electron microscopy
Sarah Stanley talks to EMBL scientists about cryo-EM. Click on the image to play.
Duration: 7 mins.
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