The hepatitis C virus internal ribosome entry site adopts an ion-dependent tertiary fold

被引:193
作者
Kieft, JS
Zhou, KH
Jubin, R
Murray, MG
Lau, JYN
Doudna, JA
机构
[1] Yale Univ, Dept Mol Biophys & Biochem, New Haven, CT 06520 USA
[2] Yale Univ, Howard Hughes Med Inst, New Haven, CT 06520 USA
[3] Schering Plough Corp, Inst Res, Kenilworth, NJ 07033 USA
关键词
RNA structure; translation; internal ribosomal entry site (IRES); hepatitis C virus (HCV); chemical and enzymatic probing;
D O I
10.1006/jmbi.1999.3095
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) located in the 5' untranslated region of the genomic RNA that drives cap-independent initiation of translation of the viral message. The approximate secondary structure and minimum functional length of the HCV IRES are known, and extensive mutagenesis has established that nearly all secondary structural domains are critical for activity. However, the presence of an IRES RNA tertiary fold and its functional relevance have not been established. Using chemical and enzymatic probes of the HCV IRES RNA in solution, we show that the IRES adopts a unique three-dimensional structure at physiological salt concentrations in the absence of additional cofactors or the translation apparatus. Folding of the IRES involves cooperative uptake of magnesium and is driven primarily by charge neutralization. This tertiary structure contains at least two independently folded regions which closely correspond to putative binding sites for the 40 S ribosomal subunit and initiation factor 3 (eIF3). Point mutations that inhibit IRES folding also inhibit its function, suggesting that the IRES tertiary structure is essential for translation initiation activity. Chemical and enzymatic probing data and small-angle X-ray scattering (SAXS) experiments in solution show that upon folding, the IRES forms an extended structure in which functionally important loops are exposed. These results suggest that the 40 S ribosomal subunit and eIF3 bind an HCV IRES that is prefolded to spatially organize recognition domains. (C) 1999 Academic Press.
引用
收藏
页码:513 / 529
页数:17
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