EMD-21133
Cryo-EM SPA : Structure of the PCBP2/Stem Loop IVm complex underlying the functionality of the poliovirus type I IRES.
EMD-21133
Single-particle6.1 Å
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Map released: 12/08/2020
Last modified: 26/08/2020
Sample Organism:
Poliovirus type 1 (strain Mahoney),
Homo sapiens
Sample: truncated stem loop IV of the poliovirus IRES, residues, 278-398 in complex with PolyC binding protein 2
Deposition Authors: Wilce MCJ, Wilce JA
Sample: truncated stem loop IV of the poliovirus IRES, residues, 278-398 in complex with PolyC binding protein 2
Deposition Authors: Wilce MCJ, Wilce JA
Structure of the PCBP2/stem-loop IV complex underlying translation initiation mediated by the poliovirus type I IRES.
Beckham SA,
Matak MY,
Belousoff MJ
,
Venugopal H
,
Shah N
,
Vankadari N
,
Elmlund H
,
Nguyen JHC,
Semler BL,
Wilce MCJ,
Wilce JA
(2020) Nucleic Acids Res. , 48 , 8006 - 8021
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(2020) Nucleic Acids Res. , 48 , 8006 - 8021
Abstract:
The poliovirus type I IRES is able to recruit ribosomal machinery only in the presence of host factor PCBP2 that binds to stem-loop IV of the IRES. When PCBP2 is cleaved in its linker region by viral proteinase 3CD, translation initiation ceases allowing the next stage of replication to commence. Here, we investigate the interaction of PCBP2 with the apical region of stem-loop IV (SLIVm) of poliovirus RNA in its full-length and truncated form. CryoEM structure reconstruction of the full-length PCBP2 in complex with SLIVm solved to 6.1 Å resolution reveals a compact globular complex of PCBP2 interacting with the cruciform RNA via KH domains and featuring a prominent GNRA tetraloop. SEC-SAXS, SHAPE and hydroxyl-radical cleavage establish that PCBP2 stabilizes the SLIVm structure, but upon cleavage in the linker domain the complex becomes more flexible and base accessible. Limited proteolysis and REMSA demonstrate the accessibility of the linker region in the PCBP2/SLIVm complex and consequent loss of affinity of PCBP2 for the SLIVm upon cleavage. Together this study sheds light on the structural features of the PCBP2/SLIV complex vital for ribosomal docking, and the way in which this key functional interaction is regulated following translation of the poliovirus genome.
The poliovirus type I IRES is able to recruit ribosomal machinery only in the presence of host factor PCBP2 that binds to stem-loop IV of the IRES. When PCBP2 is cleaved in its linker region by viral proteinase 3CD, translation initiation ceases allowing the next stage of replication to commence. Here, we investigate the interaction of PCBP2 with the apical region of stem-loop IV (SLIVm) of poliovirus RNA in its full-length and truncated form. CryoEM structure reconstruction of the full-length PCBP2 in complex with SLIVm solved to 6.1 Å resolution reveals a compact globular complex of PCBP2 interacting with the cruciform RNA via KH domains and featuring a prominent GNRA tetraloop. SEC-SAXS, SHAPE and hydroxyl-radical cleavage establish that PCBP2 stabilizes the SLIVm structure, but upon cleavage in the linker domain the complex becomes more flexible and base accessible. Limited proteolysis and REMSA demonstrate the accessibility of the linker region in the PCBP2/SLIVm complex and consequent loss of affinity of PCBP2 for the SLIVm upon cleavage. Together this study sheds light on the structural features of the PCBP2/SLIV complex vital for ribosomal docking, and the way in which this key functional interaction is regulated following translation of the poliovirus genome.