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Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts
Author(s) -
Tesina Petr,
Lessen Laura N,
Buschauer Robert,
Cheng Jingdong,
Wu Colin ChihChien,
Berninghausen Otto,
Buskirk Allen R,
Becker Thomas,
Beckmann Roland,
Green Rachel
Publication year - 2019
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.15252/embj.2019103365
Subject(s) - library science , center (category theory) , chemistry , computer science , crystallography
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.