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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 by 2D PAGE of proteins cross-linked to 32P-labeled HCV IRES derivatives in the binary complexes with the 40S subunits. (a–c), The autoradiograms correspond to the experiments with HCV IRES derivatives containing the cross-linker at nucleotide A275, G263 or C83 (marked in the respective panels). (d), Coomassie stained gel corresponding to (b) (as example); the positions of the proteins are indicated (44, 45). The locations of the radioactive spots corresponding to the cross-linked proteins are indicated on the stained gel by dotted lines. The cross-linked proteins are highlighted by an asterisk (*), and are also in bold in (d).
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Figure 5: Analysis by 2D PAGE of proteins cross-linked to 32P-labeled HCV IRES derivatives in the binary complexes with the 40S subunits. (a–c), The autoradiograms correspond to the experiments with HCV IRES derivatives containing the cross-linker at nucleotide A275, G263 or C83 (marked in the respective panels). (d), Coomassie stained gel corresponding to (b) (as example); the positions of the proteins are indicated (44, 45). The locations of the radioactive spots corresponding to the cross-linked proteins are indicated on the stained gel by dotted lines. The cross-linked proteins are highlighted by an asterisk (*), and are also in bold in (d).

Mentions: Cross-linked protein(s) were more precisely identified resolving them after exhaustive hydrolysis of HCV IRES with RNAses A and T1 by ‘basic-SDS’ 2D PAGE (separation in the first dimension was at pH 8.3, and in the second dimension at pH 6.75 in the presence of SDS), the results are presented in Figure 5. Cross-linked proteins were identified considering that in the electrophoretic system used, oligoribonucleotide fragments cross-linked to proteins decrease their mobilities in the first dimension but barely affect their migration in the second dimension. Consequently, the radioactive spot of the cross-linked protein should be somewhat shifted to the left of the corresponding stained spot of the unmodified protein. The extents of the shifts were evaluated based on data of our previous studies on cross-linking of short mRNA analogues to ribosomes (25,26). The results showed that HCV IRES derivatized at the A275 and the G263 cross-linked to the same three groups of proteins, namely, to S3/S3a (less probably, S2), S10/S15/S14 and S13/S16 (much less probably, S25), the distribution of cross-links between these groups depended on the location of the cross-linker in the HCV IRES (Figure 5a and b). With C83-modified IRES, only two spots corresponding to cross-linked proteins S10/S15/S14 and S13/S16 were detected (Figure 5c). Given the results of the 1D gels (Figure 4), more exact identification of the cross-linked proteins could be performed. In the group S13/S16/S25, only protein S16 was actually cross-linked since S13 and S25 were not among the candidate cross-linked proteins (see above). For the same reason, proteins S10 and S15 could be excluded from the group S10/S15/S14 and the only cross-linked protein was S14. Similarly, protein S3a could be identified as the only cross-linked protein in the group S3/S3a. Thus, combining the data of 1D and 2D PAGE made it possible to conclude that all HCV IRES derivatives used here cross-linked mainly to proteins S14 and S16, and the derivatives bearing the modifying group at A275 or G263 also cross-linked to protein S3a (Figure 5d).Figure 5.


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 by 2D PAGE of proteins cross-linked to 32P-labeled HCV IRES derivatives in the binary complexes with the 40S subunits. (a–c), The autoradiograms correspond to the experiments with HCV IRES derivatives containing the cross-linker at nucleotide A275, G263 or C83 (marked in the respective panels). (d), Coomassie stained gel corresponding to (b) (as example); the positions of the proteins are indicated (44, 45). The locations of the radioactive spots corresponding to the cross-linked proteins are indicated on the stained gel by dotted lines. The cross-linked proteins are highlighted by an asterisk (*), and are also in bold in (d).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Analysis by 2D PAGE of proteins cross-linked to 32P-labeled HCV IRES derivatives in the binary complexes with the 40S subunits. (a–c), The autoradiograms correspond to the experiments with HCV IRES derivatives containing the cross-linker at nucleotide A275, G263 or C83 (marked in the respective panels). (d), Coomassie stained gel corresponding to (b) (as example); the positions of the proteins are indicated (44, 45). The locations of the radioactive spots corresponding to the cross-linked proteins are indicated on the stained gel by dotted lines. The cross-linked proteins are highlighted by an asterisk (*), and are also in bold in (d).
Mentions: Cross-linked protein(s) were more precisely identified resolving them after exhaustive hydrolysis of HCV IRES with RNAses A and T1 by ‘basic-SDS’ 2D PAGE (separation in the first dimension was at pH 8.3, and in the second dimension at pH 6.75 in the presence of SDS), the results are presented in Figure 5. Cross-linked proteins were identified considering that in the electrophoretic system used, oligoribonucleotide fragments cross-linked to proteins decrease their mobilities in the first dimension but barely affect their migration in the second dimension. Consequently, the radioactive spot of the cross-linked protein should be somewhat shifted to the left of the corresponding stained spot of the unmodified protein. The extents of the shifts were evaluated based on data of our previous studies on cross-linking of short mRNA analogues to ribosomes (25,26). The results showed that HCV IRES derivatized at the A275 and the G263 cross-linked to the same three groups of proteins, namely, to S3/S3a (less probably, S2), S10/S15/S14 and S13/S16 (much less probably, S25), the distribution of cross-links between these groups depended on the location of the cross-linker in the HCV IRES (Figure 5a and b). With C83-modified IRES, only two spots corresponding to cross-linked proteins S10/S15/S14 and S13/S16 were detected (Figure 5c). Given the results of the 1D gels (Figure 4), more exact identification of the cross-linked proteins could be performed. In the group S13/S16/S25, only protein S16 was actually cross-linked since S13 and S25 were not among the candidate cross-linked proteins (see above). For the same reason, proteins S10 and S15 could be excluded from the group S10/S15/S14 and the only cross-linked protein was S14. Similarly, protein S3a could be identified as the only cross-linked protein in the group S3/S3a. Thus, combining the data of 1D and 2D PAGE made it possible to conclude that all HCV IRES derivatives used here cross-linked mainly to proteins S14 and S16, and the derivatives bearing the modifying group at A275 or G263 also cross-linked to protein S3a (Figure 5d).Figure 5.

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