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Positioning of subdomain IIId and apical loop of domain II of the hepatitis C IRES on the human 40S ribosome.

Babaylova E, Graifer D, Malygin A, Stahl J, Shatsky I, Karpova G - Nucleic Acids Res. (2009)

Bottom Line: The 5'-untranslated region of the hepatitis C virus (HCV) RNA contains a highly structured motif called IRES (Internal Ribosome Entry Site) responsible for the cap-independent initiation of the viral RNA translation.At first, the IRES binds to the 40S subunit without any initiation factors so that the initiation AUG codon falls into the P site.HCV IRES derivatives that bear a photoactivatable group at nucleotide A275 or at G263 in subdomain IIId cross-link to ribosomal proteins S3a, S14 and S16, and HCV IRES derivatized at the C83 in the apex of domain II cross-link to proteins S14 and S16.

View Article: PubMed Central - PubMed

Affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia.

ABSTRACT
The 5'-untranslated region of the hepatitis C virus (HCV) RNA contains a highly structured motif called IRES (Internal Ribosome Entry Site) responsible for the cap-independent initiation of the viral RNA translation. At first, the IRES binds to the 40S subunit without any initiation factors so that the initiation AUG codon falls into the P site. Here using an original site-directed cross-linking strategy, we identified 40S subunit components neighboring subdomain IIId, which is critical for HCV IRES binding to the subunit, and apical loop of domain II, which was suggested to contact the 40S subunit from data on cryo-electron microscopy of ribosomal complexes containing the HCV IRES. HCV IRES derivatives that bear a photoactivatable group at nucleotide A275 or at G263 in subdomain IIId cross-link to ribosomal proteins S3a, S14 and S16, and HCV IRES derivatized at the C83 in the apex of domain II cross-link to proteins S14 and S16.

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Scheme of site-specific introduction of a photoactivatable group into specific RNA sites based on the site-specific alkylation of the RNA with [4-(N-2-chloroethyl-N-methylamino)benzyl]-phosphoramides of oligodeoxyribonucleotides. (a) Order of chemical reactions. (b) Nucleotide sequence of HCV IRES. Target sequences for the deoxy-oligomer derivatives are given in bold, the respective complementary oligodeoxyribonucleotides are shown with either gray or black lines under the sequence, and the letters ‘p’ indicate the terminal 5′-phosphates derivatized with alkylating groups. Helper oligomers are shown by dotted lines. Lines with arrows above the sequence indicate primers used for reverse transcription (arrows show the direction of primer extension). RNA sequences complementary to the primers are given in italics. Either gray or black vertical arrows show cross-linked nucleotides.
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Figure 2: Scheme of site-specific introduction of a photoactivatable group into specific RNA sites based on the site-specific alkylation of the RNA with [4-(N-2-chloroethyl-N-methylamino)benzyl]-phosphoramides of oligodeoxyribonucleotides. (a) Order of chemical reactions. (b) Nucleotide sequence of HCV IRES. Target sequences for the deoxy-oligomer derivatives are given in bold, the respective complementary oligodeoxyribonucleotides are shown with either gray or black lines under the sequence, and the letters ‘p’ indicate the terminal 5′-phosphates derivatized with alkylating groups. Helper oligomers are shown by dotted lines. Lines with arrows above the sequence indicate primers used for reverse transcription (arrows show the direction of primer extension). RNA sequences complementary to the primers are given in italics. Either gray or black vertical arrows show cross-linked nucleotides.

Mentions: To introduce photoactivatable perfluorophenyl azide groups into hairpin IIId or the apex of domain II of the IRES (Figure 1), an approach based on the site-specific alkylation of RNA with phosphoramide derivatives of deoxyribonucleotides described previously (28) and schematically shown in Figure 2a was applied. To increase the yield of IRES alkylation with the derivatives of deoxy-oligomers complementary to sequences 259–276 and 62–81, helper oligomers were used that facilitate binding of oligomers bearing the alkylating group to the structured RNA [see (34) and Refs. therein] (Figures 1 and 2b). Helpers were not used in the experiments with the derivative of oligomer complementary to sequence 248–267 since the yield of HCV IRES alkylation was sufficiently high. The covalent adducts resulting from IRES alkylation with the oligonucleotide derivatives were separated from unmodified RNA by denaturing PAGE on an 8% gel (the respective electrophoregrams are not shown). Generally, about 50% of the RNA was converted into the covalent adduct in the course of alkylation.Figure 2.


Positioning of subdomain IIId and apical loop of domain II of the hepatitis C IRES on the human 40S ribosome.

Babaylova E, Graifer D, Malygin A, Stahl J, Shatsky I, Karpova G - Nucleic Acids Res. (2009)

Scheme of site-specific introduction of a photoactivatable group into specific RNA sites based on the site-specific alkylation of the RNA with [4-(N-2-chloroethyl-N-methylamino)benzyl]-phosphoramides of oligodeoxyribonucleotides. (a) Order of chemical reactions. (b) Nucleotide sequence of HCV IRES. Target sequences for the deoxy-oligomer derivatives are given in bold, the respective complementary oligodeoxyribonucleotides are shown with either gray or black lines under the sequence, and the letters ‘p’ indicate the terminal 5′-phosphates derivatized with alkylating groups. Helper oligomers are shown by dotted lines. Lines with arrows above the sequence indicate primers used for reverse transcription (arrows show the direction of primer extension). RNA sequences complementary to the primers are given in italics. Either gray or black vertical arrows show cross-linked nucleotides.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Scheme of site-specific introduction of a photoactivatable group into specific RNA sites based on the site-specific alkylation of the RNA with [4-(N-2-chloroethyl-N-methylamino)benzyl]-phosphoramides of oligodeoxyribonucleotides. (a) Order of chemical reactions. (b) Nucleotide sequence of HCV IRES. Target sequences for the deoxy-oligomer derivatives are given in bold, the respective complementary oligodeoxyribonucleotides are shown with either gray or black lines under the sequence, and the letters ‘p’ indicate the terminal 5′-phosphates derivatized with alkylating groups. Helper oligomers are shown by dotted lines. Lines with arrows above the sequence indicate primers used for reverse transcription (arrows show the direction of primer extension). RNA sequences complementary to the primers are given in italics. Either gray or black vertical arrows show cross-linked nucleotides.
Mentions: To introduce photoactivatable perfluorophenyl azide groups into hairpin IIId or the apex of domain II of the IRES (Figure 1), an approach based on the site-specific alkylation of RNA with phosphoramide derivatives of deoxyribonucleotides described previously (28) and schematically shown in Figure 2a was applied. To increase the yield of IRES alkylation with the derivatives of deoxy-oligomers complementary to sequences 259–276 and 62–81, helper oligomers were used that facilitate binding of oligomers bearing the alkylating group to the structured RNA [see (34) and Refs. therein] (Figures 1 and 2b). Helpers were not used in the experiments with the derivative of oligomer complementary to sequence 248–267 since the yield of HCV IRES alkylation was sufficiently high. The covalent adducts resulting from IRES alkylation with the oligonucleotide derivatives were separated from unmodified RNA by denaturing PAGE on an 8% gel (the respective electrophoregrams are not shown). Generally, about 50% of the RNA was converted into the covalent adduct in the course of alkylation.Figure 2.

Bottom Line: The 5'-untranslated region of the hepatitis C virus (HCV) RNA contains a highly structured motif called IRES (Internal Ribosome Entry Site) responsible for the cap-independent initiation of the viral RNA translation.At first, the IRES binds to the 40S subunit without any initiation factors so that the initiation AUG codon falls into the P site.HCV IRES derivatives that bear a photoactivatable group at nucleotide A275 or at G263 in subdomain IIId cross-link to ribosomal proteins S3a, S14 and S16, and HCV IRES derivatized at the C83 in the apex of domain II cross-link to proteins S14 and S16.

View Article: PubMed Central - PubMed

Affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia.

ABSTRACT
The 5'-untranslated region of the hepatitis C virus (HCV) RNA contains a highly structured motif called IRES (Internal Ribosome Entry Site) responsible for the cap-independent initiation of the viral RNA translation. At first, the IRES binds to the 40S subunit without any initiation factors so that the initiation AUG codon falls into the P site. Here using an original site-directed cross-linking strategy, we identified 40S subunit components neighboring subdomain IIId, which is critical for HCV IRES binding to the subunit, and apical loop of domain II, which was suggested to contact the 40S subunit from data on cryo-electron microscopy of ribosomal complexes containing the HCV IRES. HCV IRES derivatives that bear a photoactivatable group at nucleotide A275 or at G263 in subdomain IIId cross-link to ribosomal proteins S3a, S14 and S16, and HCV IRES derivatized at the C83 in the apex of domain II cross-link to proteins S14 and S16.

Show MeSH
Related in: MedlinePlus