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.

Central European Institute of Technology Masaryk University Kamenice 5 62500 Brno Czech Republic

Department of Physical Biochemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany

Department of Theoretical and Computational Biophysics Max Planck Institute for Biophysical Chemistry Am Fassberg 11 Göttingen 37077 Germany

Gene Center Department for Biochemistry and Center for integrated Protein Science Munich University of Munich Feodor Lynenstr 25 81377 Munich Germany

Gene Center Department for Biochemistry and Center for integrated Protein Science Munich University of Munich Feodor Lynenstr 25 81377 Munich Germany; Institute for Biochemistry and Molecular Biology University of Hamburg Martin Luther King Platz 6 20146 Hamburg Germany

University of Tartu Institute of Technology Nooruse 1 50411 Tartu Estonia

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Binding of the peptide deformylase on the ribosome surface modulates the exit tunnel interior