Abstract
Eukaryotic protein synthesis begins with mRNA positioning in the ribosomal decoding channel in a process typically controlled by translation-initiation factors. Some viruses use an internal ribosome entry site (IRES) in their mRNA to harness ribosomes independently of initiation factors. We show here that a ribosome conformational change that is induced upon hepatitis C viral IRES binding is necessary but not sufficient for correct mRNA positioning. Using directed hydroxyl radical probing to monitor the assembly of IRES-containing translation-initiation complexes, we have defined a crucial step in which mRNA is stabilized upon initiator tRNA binding. Unexpectedly, however, this stabilization occurs independently of the AUG codon, underscoring the importance of initiation factor–mediated interactions that influence the configuration of the decoding channel. These results reveal how an IRES RNA supplants some, but not all, of the functions normally carried out by protein factors during initiation of protein synthesis.
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Acknowledgements
We thank D. King at the University of California, Berkeley, for expert MS analysis of modified proteins. We gratefully acknowledge members of the Doudna laboratory for discussions and comments on the manuscript. In particular, we would like to thank R. Spanggord for advice on hydroxyl radical probing and F. Siu for advice on RNA transcription protocols. This work was supported in part by a grant from the US National Institutes of Health to J.A.D. and J.W.B.H.
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C.S.F. performed the experiments; C.S.F., J.W.B.H. and J.A.D. designed experiments and wrote the manuscript.
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Fraser, C., Hershey, J. & Doudna, J. The pathway of hepatitis C virus mRNA recruitment to the human ribosome. Nat Struct Mol Biol 16, 397–404 (2009). https://doi.org/10.1038/nsmb.1572
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DOI: https://doi.org/10.1038/nsmb.1572
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