@article{6959cc61c6e84c59b37b4b95457ec051,
title = "Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts",
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- and collision-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 a novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.",
keywords = "disome, frameshift, ribosome collision, stalling, translation",
author = "Petr Tesina and Lessen, {Laura N.} and Robert Buschauer and Jingdong Cheng and Wu, {Colin Chih Chien} and Otto Berninghausen and Buskirk, {Allen R.} and Thomas Becker and Roland Beckmann and Rachel Green",
note = "Funding Information: The authors would like to thank Elisabeth Heckel for help with sample preparation and Charlotte Ungewickell and Susanne Rieder for technical support. The authors would also like to thank members of the Green Lab for helpful discussions, Elizabeth Grayhack for the tRNA and synthetase plasmids, and David Mohr and the Johns Hopkins Genetic Resources Core Facility for sequencing assistance. This study was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG) (DFG; BE1814/15-1) to RBe and a Ph.D. fellowship from Boehringer Ingelheim Fonds (BIF) to RBu. Additionally, this work was supported by the NIH (2R37GM059425 to RG), T32 GM008403, and HHMI (RG and CW). Funding Information: The authors would like to thank Elisabeth Heckel for help with sample preparation and Charlotte Ungewickell and Susanne Rieder for technical support. The authors would also like to thank members of the Green Lab for helpful discussions, Elizabeth Grayhack for the tRNA and synthetase plasmids, and David Mohr and the Johns Hopkins Genetic Resources Core Facility for sequencing assistance. This study was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG) (DFG; BE1814/15‐1) to RBe and a Ph.D. fellowship from Boehringer Ingelheim Fonds (BIF) to RBu. Additionally, this work was supported by the NIH (2R37GM059425 to RG), T32 GM008403, and HHMI (RG and CW). Publisher Copyright: {\textcopyright} 2019 The Authors",
year = "2020",
month = feb,
day = "3",
doi = "10.15252/embj.2019103365",
language = "English (US)",
volume = "39",
journal = "EMBO Journal",
issn = "0261-4189",
publisher = "Nature Publishing Group",
number = "3",
}