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Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts

View ORCID ProfilePetr Tesina, Laura N. Lessen, Robert Buschauer, Jingdong Cheng, Colin Chih-Chien Wu, Otto Berninghausen, Allen R. Buskirk, Thomas Becker, Roland Beckmann, Rachel Green
doi: https://doi.org/10.1101/755652
Petr Tesina
1Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany
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  • ORCID record for Petr Tesina
Laura N. Lessen
2Program in Molecular Biophysics, Johns Hopkins University School of Medicine, Baltimore, MD 21205; Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205
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Robert Buschauer
1Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany
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Jingdong Cheng
1Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany
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Colin Chih-Chien Wu
2Program in Molecular Biophysics, Johns Hopkins University School of Medicine, Baltimore, MD 21205; Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205
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Otto Berninghausen
1Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany
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Allen R. Buskirk
3Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205
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Thomas Becker
1Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany
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  • For correspondence: becker@genzentrum.lmu.de beckmann@genzentrum.lmu.de ragreen@jhmi.edu
Roland Beckmann
1Gene Center and Center for Integrated Protein Science Munich, Department of Biochemistry, University of Munich, Munich, Germany
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  • For correspondence: becker@genzentrum.lmu.de beckmann@genzentrum.lmu.de ragreen@jhmi.edu
Rachel Green
3Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205
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  • For correspondence: becker@genzentrum.lmu.de beckmann@genzentrum.lmu.de ragreen@jhmi.edu
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Abstract

Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of in vitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an in vitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.

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Posted September 04, 2019.
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Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts
Petr Tesina, Laura N. Lessen, Robert Buschauer, Jingdong Cheng, Colin Chih-Chien Wu, Otto Berninghausen, Allen R. Buskirk, Thomas Becker, Roland Beckmann, Rachel Green
bioRxiv 755652; doi: https://doi.org/10.1101/755652
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Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts
Petr Tesina, Laura N. Lessen, Robert Buschauer, Jingdong Cheng, Colin Chih-Chien Wu, Otto Berninghausen, Allen R. Buskirk, Thomas Becker, Roland Beckmann, Rachel Green
bioRxiv 755652; doi: https://doi.org/10.1101/755652

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