mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding [preprint]
Bao, Chen ; Loerch, Sarah ; Ling, Clarence ; Korostelev, Andrei A. ; Grigorieff, Nikolaus ; Ermolenko, Dmitri N.
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mRNA
ribosomes
tRNA binding
frameshift-inducing stem-loops
helicases
Förster resonance energy transfer
E. coli dnaX
Human Immunodeficiency Virus
Biochemical Phenomena, Metabolism, and Nutrition
Biochemistry
Enzymes and Coenzymes
Genetic Phenomena
Nucleic Acids, Nucleotides, and Nucleosides
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Abstract
Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.
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bioRxiv 2020.02.05.936120; doi: https://doi.org/10.1101/2020.02.05.936120. Link to preprint on bioRxiv service.
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Now published in eLife doi: 10.7554/eLife.55799