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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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Analysis of 40S proteins cross-linked to 32P-labeled HCV IRES derivatives (nucleotides bearing the cross-linker are indicated at the top) by immunoprecipitation using antibodies against mammalian 40S ribosomal proteins (indicated at the bottom).
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Figure 6: Analysis of 40S proteins cross-linked to 32P-labeled HCV IRES derivatives (nucleotides bearing the cross-linker are indicated at the top) by immunoprecipitation using antibodies against mammalian 40S ribosomal proteins (indicated at the bottom).

Mentions: The identity of the cross-linked proteins S3a, S14 and S16 was confirmed by immunoprecipitation using specific antibodies. In parallel, immunoprecipitation with antibodies against proteins S2 and S3 whose modification was practically excluded on the basis of the comparison of the results of 1D and 2D PAGE analysis was carried out as control. The results shown in Figure 6 are in a good accordance with the data of 1D and 2D PAGE separations discussed above (Figure 5) and confirm cross-linking of the HCV IRES derivatives to ribosomal proteins S3a, S14 and S16.Figure 6.


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)

Analysis of 40S proteins cross-linked to 32P-labeled HCV IRES derivatives (nucleotides bearing the cross-linker are indicated at the top) by immunoprecipitation using antibodies against mammalian 40S ribosomal proteins (indicated at the bottom).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651777&req=5

Figure 6: Analysis of 40S proteins cross-linked to 32P-labeled HCV IRES derivatives (nucleotides bearing the cross-linker are indicated at the top) by immunoprecipitation using antibodies against mammalian 40S ribosomal proteins (indicated at the bottom).
Mentions: The identity of the cross-linked proteins S3a, S14 and S16 was confirmed by immunoprecipitation using specific antibodies. In parallel, immunoprecipitation with antibodies against proteins S2 and S3 whose modification was practically excluded on the basis of the comparison of the results of 1D and 2D PAGE analysis was carried out as control. The results shown in Figure 6 are in a good accordance with the data of 1D and 2D PAGE separations discussed above (Figure 5) and confirm cross-linking of the HCV IRES derivatives to ribosomal proteins S3a, S14 and S16.Figure 6.

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