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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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Identification of sites of cross-linking of oligonucleotide derivatives to the HCV IRES by reverse transcription and binding properties of the HCV IRES derivatives. (a) Extension of [5′-32P]-labeled primers complementary to the HCV IRES sequences 331–350 (left panel) and 103–120 (right panel). Lanes 1, 2 and 3, primer extension on HCV IRES alkylated with derivatives of deoxy-oligomers complementary to the sequences 259–276, 248–267 and 62–81, respectively. Lanes K, primer extension with control HCV IRES incubated under conditions of alkylation but without oligomer derivatives. Lanes U, G, C, A, sequencing of HCV IRES. Arrows indicate positions of the reverse transcription stops caused by the cross-links. (b) Isotherms of binding of control unmodified HCV IRES (K) and its derivatives containing a perfluorophenyl azide group at A275, G263 or C83 to 40S subunits (1, 2 and 3, respectively). The initial concentration of the HCV IRES or its derivatives was 1.0 × 10−7 M. Relative error was about 10%.
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Figure 3: Identification of sites of cross-linking of oligonucleotide derivatives to the HCV IRES by reverse transcription and binding properties of the HCV IRES derivatives. (a) Extension of [5′-32P]-labeled primers complementary to the HCV IRES sequences 331–350 (left panel) and 103–120 (right panel). Lanes 1, 2 and 3, primer extension on HCV IRES alkylated with derivatives of deoxy-oligomers complementary to the sequences 259–276, 248–267 and 62–81, respectively. Lanes K, primer extension with control HCV IRES incubated under conditions of alkylation but without oligomer derivatives. Lanes U, G, C, A, sequencing of HCV IRES. Arrows indicate positions of the reverse transcription stops caused by the cross-links. (b) Isotherms of binding of control unmodified HCV IRES (K) and its derivatives containing a perfluorophenyl azide group at A275, G263 or C83 to 40S subunits (1, 2 and 3, respectively). The initial concentration of the HCV IRES or its derivatives was 1.0 × 10−7 M. Relative error was about 10%.

Mentions: To identify modified IRES nucleotides, reverse transcription was used that makes it possible to detect modified nucleotides by stop or pause of primer extension. The modification site is generally assumed to be the nucleotide 5′ of the primer extension stop site. Therefore, stops at A276 (Figure 3a, left panel, lane 1), U264 (left panel, lane 2) and C84 (right panel, lane 3) correspond to modified A275, G263 and C83, respectively (Figures 1 and 2b). With HCV IRES alkylated with the derivative of oligomer complementary to sequence 248–267, a weaker stop at G263 was also observed (Figure 3a, left panel, lane 2). This signal could not be assigned to the primer extension stop caused by the modification of U262 since uridine is unable to react with aromatic 2-chloroethyl amines at nearly neutral pH (35). Thus, the signal at nucleotide in position 263 was assigned to a pause of reverse transcription that could occur at guanine alkylated on the N7 atom, which is not involved in Watson–Crick base pairing. One could expect that in the heteroduplex of the HCV IRES with the derivative of oligomer complementary to sequence 248–267 the alkylating group is located closer to nucleotides G265–267 rather than to G263 (Figure 1). The unusual target of alkylation in this case might be due to peculiarities of the spatial structure of the IRES and also to the low reactivity of guanines in oligoG fragments towards alkylation with aromatic 2-chloroethyl amines (36). Weaker stops with the derivative of oligomer complementary to sequence 62–81 probably correspond to minor alkylation sites adjacent to C83 (taking into account the discussion above, these are G82 and A81).Figure 3.


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)

Identification of sites of cross-linking of oligonucleotide derivatives to the HCV IRES by reverse transcription and binding properties of the HCV IRES derivatives. (a) Extension of [5′-32P]-labeled primers complementary to the HCV IRES sequences 331–350 (left panel) and 103–120 (right panel). Lanes 1, 2 and 3, primer extension on HCV IRES alkylated with derivatives of deoxy-oligomers complementary to the sequences 259–276, 248–267 and 62–81, respectively. Lanes K, primer extension with control HCV IRES incubated under conditions of alkylation but without oligomer derivatives. Lanes U, G, C, A, sequencing of HCV IRES. Arrows indicate positions of the reverse transcription stops caused by the cross-links. (b) Isotherms of binding of control unmodified HCV IRES (K) and its derivatives containing a perfluorophenyl azide group at A275, G263 or C83 to 40S subunits (1, 2 and 3, respectively). The initial concentration of the HCV IRES or its derivatives was 1.0 × 10−7 M. Relative error was about 10%.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: Identification of sites of cross-linking of oligonucleotide derivatives to the HCV IRES by reverse transcription and binding properties of the HCV IRES derivatives. (a) Extension of [5′-32P]-labeled primers complementary to the HCV IRES sequences 331–350 (left panel) and 103–120 (right panel). Lanes 1, 2 and 3, primer extension on HCV IRES alkylated with derivatives of deoxy-oligomers complementary to the sequences 259–276, 248–267 and 62–81, respectively. Lanes K, primer extension with control HCV IRES incubated under conditions of alkylation but without oligomer derivatives. Lanes U, G, C, A, sequencing of HCV IRES. Arrows indicate positions of the reverse transcription stops caused by the cross-links. (b) Isotherms of binding of control unmodified HCV IRES (K) and its derivatives containing a perfluorophenyl azide group at A275, G263 or C83 to 40S subunits (1, 2 and 3, respectively). The initial concentration of the HCV IRES or its derivatives was 1.0 × 10−7 M. Relative error was about 10%.
Mentions: To identify modified IRES nucleotides, reverse transcription was used that makes it possible to detect modified nucleotides by stop or pause of primer extension. The modification site is generally assumed to be the nucleotide 5′ of the primer extension stop site. Therefore, stops at A276 (Figure 3a, left panel, lane 1), U264 (left panel, lane 2) and C84 (right panel, lane 3) correspond to modified A275, G263 and C83, respectively (Figures 1 and 2b). With HCV IRES alkylated with the derivative of oligomer complementary to sequence 248–267, a weaker stop at G263 was also observed (Figure 3a, left panel, lane 2). This signal could not be assigned to the primer extension stop caused by the modification of U262 since uridine is unable to react with aromatic 2-chloroethyl amines at nearly neutral pH (35). Thus, the signal at nucleotide in position 263 was assigned to a pause of reverse transcription that could occur at guanine alkylated on the N7 atom, which is not involved in Watson–Crick base pairing. One could expect that in the heteroduplex of the HCV IRES with the derivative of oligomer complementary to sequence 248–267 the alkylating group is located closer to nucleotides G265–267 rather than to G263 (Figure 1). The unusual target of alkylation in this case might be due to peculiarities of the spatial structure of the IRES and also to the low reactivity of guanines in oligoG fragments towards alkylation with aromatic 2-chloroethyl amines (36). Weaker stops with the derivative of oligomer complementary to sequence 62–81 probably correspond to minor alkylation sites adjacent to C83 (taking into account the discussion above, these are G82 and A81).Figure 3.

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