Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P

Mol Cell. 2017 Nov 2;68(3):515-527.e6. doi: 10.1016/j.molcel.2017.10.014.

Abstract

Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.

Keywords: EF-P; RNA; a-IF5A; eIF5A; nascent chain; prolines; ribosome; single particle cryo-EM; stalling; translation elongation.

MeSH terms

  • Binding Sites
  • Cryoelectron Microscopy
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Escherichia coli Proteins / ultrastructure
  • Eukaryotic Translation Initiation Factor 5A
  • Molecular Docking Simulation
  • Molecular Dynamics Simulation
  • Mutation
  • Nucleic Acid Conformation
  • Peptide Elongation Factors / chemistry
  • Peptide Elongation Factors / genetics
  • Peptide Elongation Factors / metabolism*
  • Peptide Elongation Factors / ultrastructure
  • Peptide Initiation Factors / chemistry
  • Peptide Initiation Factors / metabolism
  • Peptides / chemistry
  • Peptides / metabolism*
  • Protein Binding
  • Protein Biosynthesis
  • Protein Conformation
  • RNA, Messenger / chemistry
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Transfer / chemistry
  • RNA, Transfer / genetics
  • RNA, Transfer / metabolism
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / metabolism
  • Ribosomes / chemistry
  • Ribosomes / metabolism*
  • Ribosomes / ultrastructure
  • Structure-Activity Relationship

Substances

  • Escherichia coli Proteins
  • Peptide Elongation Factors
  • Peptide Initiation Factors
  • Peptides
  • RNA, Messenger
  • RNA-Binding Proteins
  • factor EF-P
  • polyproline
  • RNA, Transfer