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Common conformational changes induced in type 2 picornavirus IRESs by cognate trans-acting factors.

Yu Y, Abaeva IS, Marintchev A, Pestova TV, Hellen CU - Nucleic Acids Res. (2011)

Bottom Line: In addition to canonical translation factors, type 2 IRESs also require IRES trans-acting factors (ITAFs) that are hypothesized to stabilize the optimal IRES conformation that supports efficient ribosomal recruitment: the EMCV IRES is stimulated by pyrimidine tract binding protein (PTB), whereas the FMDV IRES requires PTB and ITAF(45).To test this hypothesis, we assessed the effect of ITAFs on the conformations of EMCV and FMDV IRESs by comparing their influence on hydroxyl radical cleavage of these IRESs from the central domain of eIF4G.The observed changes in cleavage patterns suggest that cognate ITAFs promote similar conformational changes that are consistent with adoption by the IRESs of comparable, more compact structures, in which domain J undergoes local conformational changes and is brought into closer proximity to the base of domain I.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, SUNY Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA.

ABSTRACT
Type 2 internal ribosomal entry sites (IRESs) of encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV) and other picornaviruses comprise five major domains H-L. Initiation of translation on these IRESs begins with specific binding of the central domain of initiation factor, eIF4G to the J-K domains, which is stimulated by eIF4A. eIF4G/eIF4A then restructure the region of ribosomal attachment on the IRES and promote recruitment of ribosomal 43S pre-initiation complexes. In addition to canonical translation factors, type 2 IRESs also require IRES trans-acting factors (ITAFs) that are hypothesized to stabilize the optimal IRES conformation that supports efficient ribosomal recruitment: the EMCV IRES is stimulated by pyrimidine tract binding protein (PTB), whereas the FMDV IRES requires PTB and ITAF(45). To test this hypothesis, we assessed the effect of ITAFs on the conformations of EMCV and FMDV IRESs by comparing their influence on hydroxyl radical cleavage of these IRESs from the central domain of eIF4G. The observed changes in cleavage patterns suggest that cognate ITAFs promote similar conformational changes that are consistent with adoption by the IRESs of comparable, more compact structures, in which domain J undergoes local conformational changes and is brought into closer proximity to the base of domain I.

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Hydroxyl radical cleavage of 18S rRNA from eIF4Gm in eIF4Gm-associated 43S complexes. (A and B) Primer extension analysis of hydroxyl radical cleavage of 18S rRNA from Fe(II)-tethered eIF4Gm in eIF4Gm-bound 43S complexes. Lanes C, T, A, G depict the corresponding 18S rRNA sequence. 18S rRNA nucleotides are indicated on the left of each panel, and cleavage sites are shown on the right. (C) Cleavages in 18S rRNA from eIF4Gm (cyan spheres) mapped onto the crystal structure of the yeast 80S ribosome (31). 18S rRNA and ribosomal proteins are shown as grey and blue ribbons. ES6 is colored orange and helices within it are numbered. The radius of the spheres is proportional to the efficiency of cleavage.
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Figure 6: Hydroxyl radical cleavage of 18S rRNA from eIF4Gm in eIF4Gm-associated 43S complexes. (A and B) Primer extension analysis of hydroxyl radical cleavage of 18S rRNA from Fe(II)-tethered eIF4Gm in eIF4Gm-bound 43S complexes. Lanes C, T, A, G depict the corresponding 18S rRNA sequence. 18S rRNA nucleotides are indicated on the left of each panel, and cleavage sites are shown on the right. (C) Cleavages in 18S rRNA from eIF4Gm (cyan spheres) mapped onto the crystal structure of the yeast 80S ribosome (31). 18S rRNA and ribosomal proteins are shown as grey and blue ribbons. ES6 is colored orange and helices within it are numbered. The radius of the spheres is proportional to the efficiency of cleavage.

Mentions: To investigate hydroxyl radical cleavage of EMCV and FMDV IRESs from the surface of eIF4Gm in IRES/eIF4Gm/eIF4A ternary complexes (Figures 1, 3 and 4), 5 pmol EMCV RNA (nucleotides 280–974) or FMDV RNA (nucleotides 280–740) were incubated at 37°C for 10 min in a total reaction volume of 50 µl containing buffer A [20 mM HEPES (pH 7.6), 100 mM KCl, 2.5 mM MgCl2 and 5% glycerol] with 10 pmol [Fe(II)-BABE]-eIF4Gm and 10 pmol eIF4A in the presence/absence of 10 pmol nPTB and ITAF45, as indicated. To investigate hydroxyl radical cleavage of EMCV from the surface of eIF4Gm in 48S complexes (Figure 4), 5 pmol EMCV RNA (nucleotides 280–974) were incubated with 10 pmol [Fe(II)-BABE]-eIF4Gm, 20 pmol eIF4A, 10 pmol 40S subunits, 15 pmol eIF2, 10 pmol eIF3, 50 pml eIF1, 50 pmol eIF1A, 20 pmol eIF4B and 15 pmol Met-tRNAiMet in the presence/absence of 10 pmol nPTB in 50 µl buffer A supplemented with 1 mM ATP, 0.2 mM Guanosine 5′-[β,γ-imido] triphosphate (GMPPNP) and 0.25 mM spermidine for 10 min at 37°C. To investigate hydroxyl radical cleavage of EMCV and FMDV IRESs from the surface of eIF4A in IRES/eIF4Gm/eIF4A ternary complexes (Figure 5), 5 pmol EMCV RNA (nucleotides 280–974) or FMDV RNA (nucleotides 280–740) were incubated at 37°C for 10 min in 50 µl buffer A with 10 pmol [Fe(II)-BABE]-eIF4A and 20 pmol eIF4G in the presence/absence of 10 pmol eIF4B, nPTB and ITAF45, as indicated. To investigate hydroxyl radical cleavage of 18S rRNA from the surface of eIF4Gm (Figure 6), 43S pre-initiation complexes containing [Fe(II)-BABE]-eIF4Gm were formed by incubating 10 pmol 40S subunits, 15 pmol eIF2, 10 pmol eIF3, 50 pmol eIF1, 50 pmol eIF1A, 15 pmol Met-tRNAiMeti, 20 pmol eIF4A and 10 pmol [Fe(II)-BABE]-eIF4Gm in 50 ml buffer A supplemented with 1 mM adenosine-5′-triphosphate (ATP), 0.2 mM Guanosine 5′-[β,γ-imido] triphosphate (GMPPNP) and 0.25 mM spermidine for 10 min at 37°C. To generate hydroxyl radicals, reaction mixtures were supplemented with 0.05% H2O2 and 5 mM ascorbic acid and incubated on ice for 10 min. Reactions were quenched by adding 20 mM thiourea. mRNAs and 18S rRNA were phenol extracted, ethanol precipitated and analyzed by primer extension using AMV reverse transcriptase and appropriate [32P]-labeled primers. cDNA products were resolved in a 6% sequencing gel.Figure 5.


Common conformational changes induced in type 2 picornavirus IRESs by cognate trans-acting factors.

Yu Y, Abaeva IS, Marintchev A, Pestova TV, Hellen CU - Nucleic Acids Res. (2011)

Hydroxyl radical cleavage of 18S rRNA from eIF4Gm in eIF4Gm-associated 43S complexes. (A and B) Primer extension analysis of hydroxyl radical cleavage of 18S rRNA from Fe(II)-tethered eIF4Gm in eIF4Gm-bound 43S complexes. Lanes C, T, A, G depict the corresponding 18S rRNA sequence. 18S rRNA nucleotides are indicated on the left of each panel, and cleavage sites are shown on the right. (C) Cleavages in 18S rRNA from eIF4Gm (cyan spheres) mapped onto the crystal structure of the yeast 80S ribosome (31). 18S rRNA and ribosomal proteins are shown as grey and blue ribbons. ES6 is colored orange and helices within it are numbered. The radius of the spheres is proportional to the efficiency of cleavage.
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Related In: Results  -  Collection

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Figure 6: Hydroxyl radical cleavage of 18S rRNA from eIF4Gm in eIF4Gm-associated 43S complexes. (A and B) Primer extension analysis of hydroxyl radical cleavage of 18S rRNA from Fe(II)-tethered eIF4Gm in eIF4Gm-bound 43S complexes. Lanes C, T, A, G depict the corresponding 18S rRNA sequence. 18S rRNA nucleotides are indicated on the left of each panel, and cleavage sites are shown on the right. (C) Cleavages in 18S rRNA from eIF4Gm (cyan spheres) mapped onto the crystal structure of the yeast 80S ribosome (31). 18S rRNA and ribosomal proteins are shown as grey and blue ribbons. ES6 is colored orange and helices within it are numbered. The radius of the spheres is proportional to the efficiency of cleavage.
Mentions: To investigate hydroxyl radical cleavage of EMCV and FMDV IRESs from the surface of eIF4Gm in IRES/eIF4Gm/eIF4A ternary complexes (Figures 1, 3 and 4), 5 pmol EMCV RNA (nucleotides 280–974) or FMDV RNA (nucleotides 280–740) were incubated at 37°C for 10 min in a total reaction volume of 50 µl containing buffer A [20 mM HEPES (pH 7.6), 100 mM KCl, 2.5 mM MgCl2 and 5% glycerol] with 10 pmol [Fe(II)-BABE]-eIF4Gm and 10 pmol eIF4A in the presence/absence of 10 pmol nPTB and ITAF45, as indicated. To investigate hydroxyl radical cleavage of EMCV from the surface of eIF4Gm in 48S complexes (Figure 4), 5 pmol EMCV RNA (nucleotides 280–974) were incubated with 10 pmol [Fe(II)-BABE]-eIF4Gm, 20 pmol eIF4A, 10 pmol 40S subunits, 15 pmol eIF2, 10 pmol eIF3, 50 pml eIF1, 50 pmol eIF1A, 20 pmol eIF4B and 15 pmol Met-tRNAiMet in the presence/absence of 10 pmol nPTB in 50 µl buffer A supplemented with 1 mM ATP, 0.2 mM Guanosine 5′-[β,γ-imido] triphosphate (GMPPNP) and 0.25 mM spermidine for 10 min at 37°C. To investigate hydroxyl radical cleavage of EMCV and FMDV IRESs from the surface of eIF4A in IRES/eIF4Gm/eIF4A ternary complexes (Figure 5), 5 pmol EMCV RNA (nucleotides 280–974) or FMDV RNA (nucleotides 280–740) were incubated at 37°C for 10 min in 50 µl buffer A with 10 pmol [Fe(II)-BABE]-eIF4A and 20 pmol eIF4G in the presence/absence of 10 pmol eIF4B, nPTB and ITAF45, as indicated. To investigate hydroxyl radical cleavage of 18S rRNA from the surface of eIF4Gm (Figure 6), 43S pre-initiation complexes containing [Fe(II)-BABE]-eIF4Gm were formed by incubating 10 pmol 40S subunits, 15 pmol eIF2, 10 pmol eIF3, 50 pmol eIF1, 50 pmol eIF1A, 15 pmol Met-tRNAiMeti, 20 pmol eIF4A and 10 pmol [Fe(II)-BABE]-eIF4Gm in 50 ml buffer A supplemented with 1 mM adenosine-5′-triphosphate (ATP), 0.2 mM Guanosine 5′-[β,γ-imido] triphosphate (GMPPNP) and 0.25 mM spermidine for 10 min at 37°C. To generate hydroxyl radicals, reaction mixtures were supplemented with 0.05% H2O2 and 5 mM ascorbic acid and incubated on ice for 10 min. Reactions were quenched by adding 20 mM thiourea. mRNAs and 18S rRNA were phenol extracted, ethanol precipitated and analyzed by primer extension using AMV reverse transcriptase and appropriate [32P]-labeled primers. cDNA products were resolved in a 6% sequencing gel.Figure 5.

Bottom Line: In addition to canonical translation factors, type 2 IRESs also require IRES trans-acting factors (ITAFs) that are hypothesized to stabilize the optimal IRES conformation that supports efficient ribosomal recruitment: the EMCV IRES is stimulated by pyrimidine tract binding protein (PTB), whereas the FMDV IRES requires PTB and ITAF(45).To test this hypothesis, we assessed the effect of ITAFs on the conformations of EMCV and FMDV IRESs by comparing their influence on hydroxyl radical cleavage of these IRESs from the central domain of eIF4G.The observed changes in cleavage patterns suggest that cognate ITAFs promote similar conformational changes that are consistent with adoption by the IRESs of comparable, more compact structures, in which domain J undergoes local conformational changes and is brought into closer proximity to the base of domain I.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, SUNY Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA.

ABSTRACT
Type 2 internal ribosomal entry sites (IRESs) of encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV) and other picornaviruses comprise five major domains H-L. Initiation of translation on these IRESs begins with specific binding of the central domain of initiation factor, eIF4G to the J-K domains, which is stimulated by eIF4A. eIF4G/eIF4A then restructure the region of ribosomal attachment on the IRES and promote recruitment of ribosomal 43S pre-initiation complexes. In addition to canonical translation factors, type 2 IRESs also require IRES trans-acting factors (ITAFs) that are hypothesized to stabilize the optimal IRES conformation that supports efficient ribosomal recruitment: the EMCV IRES is stimulated by pyrimidine tract binding protein (PTB), whereas the FMDV IRES requires PTB and ITAF(45). To test this hypothesis, we assessed the effect of ITAFs on the conformations of EMCV and FMDV IRESs by comparing their influence on hydroxyl radical cleavage of these IRESs from the central domain of eIF4G. The observed changes in cleavage patterns suggest that cognate ITAFs promote similar conformational changes that are consistent with adoption by the IRESs of comparable, more compact structures, in which domain J undergoes local conformational changes and is brought into closer proximity to the base of domain I.

Show MeSH
Related in: MedlinePlus