Submitted on June 12, 2009
Accepted on October 21, 2009

Structural Insight into Nascent Polypeptide Chain–Mediated Translational Stalling

Birgit Seidelt ¹, C. Axel Innis ², Daniel N. Wilson ¹, Marco Gartmann ¹, Jean-Paul Armache ¹, Elizabeth Villa ³, Leonardo G. Trabuco ⁴, Thomas Becker ¹, Thorsten Mielke ⁵, Klaus Schulten ⁶, Thomas A. Steitz ^7*, Roland Beckmann ^1*

¹ Gene Center and Center for integrated Protein Science Munich (CiPSM), Department for Chemistry and Biochemistry, University of Munich, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
² Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute and Yale University, New Haven, CT 06520, USA.
³ Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.; Present address: Department of Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
⁴ Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
⁵ UltraStrukturNetzwerk, Max Planck Institute for Molecular Genetics, Ihnestr. 73, 14195-Berlin, Germany and Institut für Medizinische Physik und Biophysik, Charité, Ziegelstr. 5-8, 10117 Berlin, Germany.
⁶ Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.
⁷ Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute and Yale University, New Haven, CT 06520, USA.; Department of Chemistry, Yale University, New Haven, CT 06520, USA.

^* To whom correspondence should be addressed.
Thomas A. Steitz , E-mail: thomas.steitz{at}yale.edu
Roland Beckmann , E-mail: beckmann{at}lmb.uni-muenchen.de

These authors contributed equally to this work.

Expression of the Escherichia coli tryptophanase operon depends upon ribosome stalling during translation of the upstream TnaC leader peptide, a process for which interactions between the TnaC nascent chain and the ribosomal exit tunnel are critical. We determined a 5.8 Å resolution cryo-electron microscopy and single-particle reconstruction of a ribosome stalled during translation of the tnaC leader gene. The nascent chain was extended within the exit tunnel, making contacts with ribosomal components at distinct sites. Upon stalling, two conserved residues within the peptidyltransferase center adopted conformations that preclude binding of release factors. We propose a model whereby interactions within the tunnel are relayed to the peptidyltransferase center to inhibit translation. Moreover, we show that nascent chains adopt distinct conformations within the ribosomal exit tunnel.