Mechanistic Role of Structurally Dynamic Regions in Dicistroviridae IGR IRESs |
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Authors: | Jennifer S Pfingsten Alice E Castile |
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Institution: | 1 Department of Biochemistry and Molecular Genetics, University of Colorado Denver School of Medicine, Aurora, CO 80045, USA 2 Department of Pharmacology, University of Colorado Denver School of Medicine, Aurora, CO 80045, USA 3 Howard Hughes Medical Institute, University of Colorado Denver School of Medicine, Aurora, CO 80045, USA |
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Abstract: | Dicistroviridae intergenic region (IGR) internal ribosome entry site(s) (IRES) RNAs drive a cap-independent pathway of translation initiation, recruiting both small and large ribosomal subunits to viral RNA without the use of any canonical translation initiation factors. This ability is conferred by the folded three-dimensional structure of the IRES RNA, which has been solved by X-ray crystallography. Here, we report the chemical probing of Plautia stali intestine virus IGR IRES in the unbound form, in the 40S-subunit-bound form, and in the 80S-ribosome-bound form. The results, when combined with an analysis of crystal structures, suggest that parts of the IRES RNA change structure as the preinitiation complex forms. Using mutagenesis coupled with native gel electrophoresis, preinitiation complex assembly assays, and translation initiation assays, we show that these potentially structurally dynamic elements of the IRES are involved in different steps in the pathway of ribosome recruitment and translation initiation. Like tRNAs, it appears that the IGR IRES undergoes local structural changes that are coordinated with structural changes in the ribosome, and these are critical for the IRES mechanism of action. |
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Keywords: | IGR intergenic region IRES internal ribosome entry site(s) eIF eukaryotic initiation factor RBD ribosome-binding domain PSIV Plautia stali intestine virus CrPV cricket paralysis virus cryo-EM cryo-electron microscopy PK 2 pseudoknot 2 NMIA N-methylisatoic anhydride RRL rabbit reticulocyte lysate EDTA ethylenediaminetetraacetic acid |
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