Limits...
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.

Show MeSH

Related in: MedlinePlus

Autoradiogram of the 40S ribosomal proteins cross-linked to the 32P-labeled derivatives of HCV IRES by 1D PAGE in the presence of SDS. Nucleotides bearing the cross-linker are indicated. Lane K, proteins isolated from an irradiated control complex of unmodified HCV IRES with 40S subunits. Lane TP40, silver stained gel; positions of the 40S ribosomal proteins are indicated (43,44).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2651777&req=5

Figure 4: Autoradiogram of the 40S ribosomal proteins cross-linked to the 32P-labeled derivatives of HCV IRES by 1D PAGE in the presence of SDS. Nucleotides bearing the cross-linker are indicated. Lane K, proteins isolated from an irradiated control complex of unmodified HCV IRES with 40S subunits. Lane TP40, silver stained gel; positions of the 40S ribosomal proteins are indicated (43,44).

Mentions: Ribosomal proteins cross-linked to derivatives of the IRES were first resolved by 1D SDS–PAGE. Prior to analysis, the RNA cross-linked to proteins was exhaustively hydrolyzed with RNases A and T1; ribosomal proteins were separated from labeled oligoribonucleotides resulting from hydrolysis by gel-filtration under strongly denaturing conditions (6 M guanidine chloride) to decrease possible effects related to unspecific binding of oligoribonucleotides to the proteins. The results presented in Figure 4 show that all IRES derivatives cross-linked mainly to two protein groups corresponding to the bands ‘x’ in the upper and ‘y’ in the lower parts of the gel, the intensities of the bands depending on the location of the cross-linker in the IRES. With the IRES derivatized at C83, the yield of cross-linking was significantly lower than with the two other derivatives (Figure 4). It should be also noted that in the lane corresponding to the control RNA (lane K), weak diffuse bands appeared at positions similar to those of ‘x’ and ‘y’. These bands clearly originated from a small portion of unspecific complexes of ribosomal proteins with labeled oligoribonucleotides resulting from hydrolysis of the IRES and remaining in the protein fraction after gel-filtration (see above), and not separated from the proteins even under conditions of SDS–PAGE.Figure 4.


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)

Autoradiogram of the 40S ribosomal proteins cross-linked to the 32P-labeled derivatives of HCV IRES by 1D PAGE in the presence of SDS. Nucleotides bearing the cross-linker are indicated. Lane K, proteins isolated from an irradiated control complex of unmodified HCV IRES with 40S subunits. Lane TP40, silver stained gel; positions of the 40S ribosomal proteins are indicated (43,44).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Autoradiogram of the 40S ribosomal proteins cross-linked to the 32P-labeled derivatives of HCV IRES by 1D PAGE in the presence of SDS. Nucleotides bearing the cross-linker are indicated. Lane K, proteins isolated from an irradiated control complex of unmodified HCV IRES with 40S subunits. Lane TP40, silver stained gel; positions of the 40S ribosomal proteins are indicated (43,44).
Mentions: Ribosomal proteins cross-linked to derivatives of the IRES were first resolved by 1D SDS–PAGE. Prior to analysis, the RNA cross-linked to proteins was exhaustively hydrolyzed with RNases A and T1; ribosomal proteins were separated from labeled oligoribonucleotides resulting from hydrolysis by gel-filtration under strongly denaturing conditions (6 M guanidine chloride) to decrease possible effects related to unspecific binding of oligoribonucleotides to the proteins. The results presented in Figure 4 show that all IRES derivatives cross-linked mainly to two protein groups corresponding to the bands ‘x’ in the upper and ‘y’ in the lower parts of the gel, the intensities of the bands depending on the location of the cross-linker in the IRES. With the IRES derivatized at C83, the yield of cross-linking was significantly lower than with the two other derivatives (Figure 4). It should be also noted that in the lane corresponding to the control RNA (lane K), weak diffuse bands appeared at positions similar to those of ‘x’ and ‘y’. These bands clearly originated from a small portion of unspecific complexes of ribosomal proteins with labeled oligoribonucleotides resulting from hydrolysis of the IRES and remaining in the protein fraction after gel-filtration (see above), and not separated from the proteins even under conditions of SDS–PAGE.Figure 4.

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