Published online 13 April 2006
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Proteins surrounding hairpin IIIe of the hepatitis C virus internal ribosome entry site on the human 40S ribosomal subunit
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences Novosibirsk, 630090, Russia 1 Belozersky Institute of Physico-Chemical Biology, Moscow State University Moscow, 119899, Russia
*To whom correspondence should be addressed at Institute of Chemical Biology and Fundamental Medicine, Prospekt Lavrentieva, 8, Novosibirsk, 630090, Russia. Tel: +7 383 335 62 29; Fax: +7 383 333 36 77; Email: karpova{at}niboch.nsc.ru
Received January 26, 2006. Revised February 14, 2006. Accepted March 20, 2006.
Binding of the internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA to the eIF-free 40S ribosomal subunit is the first step of initiation of translation of the viral RNA. Hairpins IIId and IIIe comprising 253–302 nt of the IRES are known to be essential for binding to the 40S subunit. Here we have examined the molecular environment of the HCV IRES in its binary complex with the human 40S ribosomal subunit. For this purpose, two RNA derivatives were used that bore a photoactivatable perfluorophenyl azide cross-linker. In one derivative the cross-linker was at the nucleotide A296 in hairpin IIIe, and in the other at G87 in domain II. Site-specific introduction of the cross-linker was performed using alkylating derivatives of oligodeoxyribonucleotides complementary to the target RNA sequences. No cross-links with the rRNA were detected with either RNA derivative. The RNA with the photoactivatable group at A296 cross-linked to proteins identified as S5 and S16 (major) and p40 and S3a (minor), while no cross-links with proteins were detected with RNA modified at G87. The results obtained indicate that hairpin IIIe is located on the solvent side of the 40S subunit head on a site opposite the beak.
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