Generation of ribosome nascent chain complexes for structural and functional studies

Research output: Contribution to journalArticle (Academic Journal)

55 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)463-71
Number of pages9
JournalJournal of Structural Biology
Volume158
Issue number3
DOIs
Publication statusPublished - Jun 2007

Keywords

  • Analytic Sample Preparation Methods
  • Cryoelectron Microscopy
  • Crystallization
  • Escherichia coli Proteins
  • Peptides
  • Protein Biosynthesis
  • Ribosomes
  • Sucrose
  • Transcription Factors

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