title: Generation of ribosome nascent chain complexes for structural and functional studies.
abstract: Biochemical and structural studies of co-translational folding, targeting and translocation depend on an efficient methodology to prepare ribosome nascent chain complexes (RNCs). Here we present our approach for the generation of homogenous and stable RNCs involving in vitro translation and affinity purification. Fusing the SecM arrest sequence, which tightly interacts with the ribosomal tunnel, to the nascent polypeptide chain significantly enhanced the stability of the RNCs. We have been able to increase the yield of the affinity purification step by engineering a tag with higher affinity. The RNCs generated with this approach have been successfully used to obtain 3D cryo-electron microscopic reconstructions of complexes with the signal recognition particle and the translocon. The established procedure is highly efficient and if scaled up could yield milligram amounts of RNCs sufficient for crystallization experiments.
authors.affiliation: ETH Zürich, Institute for Molecular Biology and Biophysics, HPK Building, Schafmattstr. 20, 8093 Zürich, Switzerland. schaffitzel@mol.biol.ethz.ch
authors.forename: Christiane
authors.initials: C
authors.lastname: Schaffitzel
authors.forename: Nenad
authors.initials: N
authors.lastname: Ban
mesh_terms: D053000:Analytic Sample Preparation Methods; D020285:Cryoelectron Microscopy; D003460:Crystallization; D029968:Escherichia coli Proteins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D014176:Protein Biosynthesis; D012270:Ribosomes / Q000737:chemistry* / Q000648:ultrastructure; D013395:Sucrose / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry*
chemical_list: D029968:Escherichia coli Proteins; D010455:Peptides; C497347:SecM protein, E coli; D014157:Transcription Factors; D013395:Sucrose