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Initiation of translation by cricket paralysis virus IRES requires its translocation in the ribosome.

Fernández IS, Bai XC, Murshudov G, Scheres SH, Ramakrishnan V - Cell (2014)

Bottom Line: By using recent advances in single-particle electron cryomicroscopy, we have solved the structure of CrPV-IRES bound to the ribosome of the yeast Kluyveromyces lactis in both the canonical and rotated states at overall resolutions of 3.7 and 3.8 Å, respectively.In both states, the pseudoknot PKI of the CrPV-IRES mimics a tRNA/mRNA interaction in the decoding center of the A site of the 40S ribosomal subunit.Translocation of the IRES by elongation factor 2 (eEF2) is required to bring the first codon of the mRNA into the A site and to allow the start of translation.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK.

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The CrPV-IRES Mimics a Pretranslocational State(A) Scheme showing the action of the ternary complex of eEF1A, tRNA, and eEF2 during the normal elongation cycle. After initiation, a cognate ternary complex binds to the empty A site and delivers an aminoacyl tRNA. After dissociation of eEF1A, this pretranslocational complex is a substrate for eEF2 binding, which stabilizes a hybrid state and catalyzes translocation of the tRNAs. Following translocation, a second round of binding of ternary complex can take place.(B) Because the ribosome-CrPV-IRES complex has PKI in the A site, it should not be able to bind eEF1A and tRNA. However, it should be able to bind eEF2, and, following translocation and dissociation of eEF2, eEF1A and tRNA should be able to bind to the now vacated A site.(C) The binding of factors eEF1A (with cognate Phe-tRNAPhe) or eEF2 to the ribosome-CrPV-IRES complex was tested both before and after translocation by copelleting experiments. The last two lanes on the gel on the left show that eEF2 with the GTP analog GDPCP can bind to the ribosome-CrPV-IRES complex, but the ternary complex of eEF1A and tRNA does not bind. However, the gel on the right shows that, after translocation by eEF2 in the presence of GTP, the ternary complex of eEF1A, tRNA, and GDPCP can bind. The presence of some residual eEF2 in this lane is due to a small fraction of untranslocated ribosomes.(D) Western blot of a gel run exactly as in (C) but probed with an antibody to the calmodulin-binding protein tag on the factors.
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fig4: The CrPV-IRES Mimics a Pretranslocational State(A) Scheme showing the action of the ternary complex of eEF1A, tRNA, and eEF2 during the normal elongation cycle. After initiation, a cognate ternary complex binds to the empty A site and delivers an aminoacyl tRNA. After dissociation of eEF1A, this pretranslocational complex is a substrate for eEF2 binding, which stabilizes a hybrid state and catalyzes translocation of the tRNAs. Following translocation, a second round of binding of ternary complex can take place.(B) Because the ribosome-CrPV-IRES complex has PKI in the A site, it should not be able to bind eEF1A and tRNA. However, it should be able to bind eEF2, and, following translocation and dissociation of eEF2, eEF1A and tRNA should be able to bind to the now vacated A site.(C) The binding of factors eEF1A (with cognate Phe-tRNAPhe) or eEF2 to the ribosome-CrPV-IRES complex was tested both before and after translocation by copelleting experiments. The last two lanes on the gel on the left show that eEF2 with the GTP analog GDPCP can bind to the ribosome-CrPV-IRES complex, but the ternary complex of eEF1A and tRNA does not bind. However, the gel on the right shows that, after translocation by eEF2 in the presence of GTP, the ternary complex of eEF1A, tRNA, and GDPCP can bind. The presence of some residual eEF2 in this lane is due to a small fraction of untranslocated ribosomes.(D) Western blot of a gel run exactly as in (C) but probed with an antibody to the calmodulin-binding protein tag on the factors.

Mentions: Elongation in eukaryotes requires the two GTPase factors eEF1A and eEF2, which are the eukaryotic homologs of EF-Tu and EF-G, respectively. Of these, eEF1A delivers incoming aminoacyl tRNAs as part of a ternary complex with GTP, and eEF2 in the GTP form binds a rotated form of the ribosome and facilitates translocation (Figure 4A). If the CrPV-IRES mimics a pretranslocation state, it should not be able to bind ternary complex initially but should be able to do so after translocation by eEF2 (Figure 4B).


Initiation of translation by cricket paralysis virus IRES requires its translocation in the ribosome.

Fernández IS, Bai XC, Murshudov G, Scheres SH, Ramakrishnan V - Cell (2014)

The CrPV-IRES Mimics a Pretranslocational State(A) Scheme showing the action of the ternary complex of eEF1A, tRNA, and eEF2 during the normal elongation cycle. After initiation, a cognate ternary complex binds to the empty A site and delivers an aminoacyl tRNA. After dissociation of eEF1A, this pretranslocational complex is a substrate for eEF2 binding, which stabilizes a hybrid state and catalyzes translocation of the tRNAs. Following translocation, a second round of binding of ternary complex can take place.(B) Because the ribosome-CrPV-IRES complex has PKI in the A site, it should not be able to bind eEF1A and tRNA. However, it should be able to bind eEF2, and, following translocation and dissociation of eEF2, eEF1A and tRNA should be able to bind to the now vacated A site.(C) The binding of factors eEF1A (with cognate Phe-tRNAPhe) or eEF2 to the ribosome-CrPV-IRES complex was tested both before and after translocation by copelleting experiments. The last two lanes on the gel on the left show that eEF2 with the GTP analog GDPCP can bind to the ribosome-CrPV-IRES complex, but the ternary complex of eEF1A and tRNA does not bind. However, the gel on the right shows that, after translocation by eEF2 in the presence of GTP, the ternary complex of eEF1A, tRNA, and GDPCP can bind. The presence of some residual eEF2 in this lane is due to a small fraction of untranslocated ribosomes.(D) Western blot of a gel run exactly as in (C) but probed with an antibody to the calmodulin-binding protein tag on the factors.
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fig4: The CrPV-IRES Mimics a Pretranslocational State(A) Scheme showing the action of the ternary complex of eEF1A, tRNA, and eEF2 during the normal elongation cycle. After initiation, a cognate ternary complex binds to the empty A site and delivers an aminoacyl tRNA. After dissociation of eEF1A, this pretranslocational complex is a substrate for eEF2 binding, which stabilizes a hybrid state and catalyzes translocation of the tRNAs. Following translocation, a second round of binding of ternary complex can take place.(B) Because the ribosome-CrPV-IRES complex has PKI in the A site, it should not be able to bind eEF1A and tRNA. However, it should be able to bind eEF2, and, following translocation and dissociation of eEF2, eEF1A and tRNA should be able to bind to the now vacated A site.(C) The binding of factors eEF1A (with cognate Phe-tRNAPhe) or eEF2 to the ribosome-CrPV-IRES complex was tested both before and after translocation by copelleting experiments. The last two lanes on the gel on the left show that eEF2 with the GTP analog GDPCP can bind to the ribosome-CrPV-IRES complex, but the ternary complex of eEF1A and tRNA does not bind. However, the gel on the right shows that, after translocation by eEF2 in the presence of GTP, the ternary complex of eEF1A, tRNA, and GDPCP can bind. The presence of some residual eEF2 in this lane is due to a small fraction of untranslocated ribosomes.(D) Western blot of a gel run exactly as in (C) but probed with an antibody to the calmodulin-binding protein tag on the factors.
Mentions: Elongation in eukaryotes requires the two GTPase factors eEF1A and eEF2, which are the eukaryotic homologs of EF-Tu and EF-G, respectively. Of these, eEF1A delivers incoming aminoacyl tRNAs as part of a ternary complex with GTP, and eEF2 in the GTP form binds a rotated form of the ribosome and facilitates translocation (Figure 4A). If the CrPV-IRES mimics a pretranslocation state, it should not be able to bind ternary complex initially but should be able to do so after translocation by eEF2 (Figure 4B).

Bottom Line: By using recent advances in single-particle electron cryomicroscopy, we have solved the structure of CrPV-IRES bound to the ribosome of the yeast Kluyveromyces lactis in both the canonical and rotated states at overall resolutions of 3.7 and 3.8 Å, respectively.In both states, the pseudoknot PKI of the CrPV-IRES mimics a tRNA/mRNA interaction in the decoding center of the A site of the 40S ribosomal subunit.Translocation of the IRES by elongation factor 2 (eEF2) is required to bring the first codon of the mRNA into the A site and to allow the start of translation.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK.

Show MeSH
Related in: MedlinePlus