Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA

Hui Zhang, James A. Endrizzi, Yi Shu, Farzin Haque, Claude Sauter, Lyudmila S. Shlyakhtenko, Yuri L Lyubchenko, Peixuan Guo, Young In Chi

Research output: Contribution to journalArticle

62 Citations (Scopus)

Abstract

The bacteriophage phi29 DNA packaging motor, one of the strongest biological motors characterized to date, is geared by a packaging RNA (pRNA) ring. When assembled from three RNA fragments, its three-way junction (3WJ) motif is highly thermostable, is resistant to 8 M urea, and remains associated at extremely low concentrations in vitro and in vivo. To elucidate the structural basis for its unusual stability, we solved the crystal structure of this pRNA 3WJ motif at 3.05 Å. The structure revealed two divalent metal ions that coordinate 4 nt of the RNA fragments. Single-molecule fluorescence resonance energy transfer (smFRET) analysis confirmed a structural change of 3WJ upon addition of Mg2+. The reported pRNA 3WJ conformation is different from a previously published construct that lacks the metal coordination sites. The phi29 DNA packaging motor contains a dodecameric connector at the vertex of the procapsid, with a central pore for DNA translocation. This portal connector serves as the foothold for pRNA binding to procapsid. Subsequent modeling of a connector/pRNA complex suggests that the pRNA of the phi29 DNA packaging motor exists as a hexameric complex serving as a sheath over the connector. The model of hexameric pRNA on the connector agrees with AFM images of the phi29 pRNA hexamer acquired in air and matches all distance parameters obtained from cross-linking, complementary modification, and chemical modification interference.

Original languageEnglish (US)
Pages (from-to)1226-1237
Number of pages12
JournalRNA
Volume19
Issue number9
DOIs
StatePublished - Sep 2013

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Product Packaging
RNA
Ions
DNA Packaging
Metals
Nucleic Acid Conformation
Fluorescence Resonance Energy Transfer
Nucleotide Motifs
Ataxia
Bacteriophages
Urea
Air
DNA

Keywords

  • Metal ions
  • RNA crystal
  • RNA nanotechnology
  • RNA therapeutics
  • Three-way junction

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Zhang, H., Endrizzi, J. A., Shu, Y., Haque, F., Sauter, C., Shlyakhtenko, L. S., ... Chi, Y. I. (2013). Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA. RNA, 19(9), 1226-1237. https://doi.org/10.1261/rna.037077.112

Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA. / Zhang, Hui; Endrizzi, James A.; Shu, Yi; Haque, Farzin; Sauter, Claude; Shlyakhtenko, Lyudmila S.; Lyubchenko, Yuri L; Guo, Peixuan; Chi, Young In.

In: RNA, Vol. 19, No. 9, 09.2013, p. 1226-1237.

Research output: Contribution to journalArticle

Zhang, H, Endrizzi, JA, Shu, Y, Haque, F, Sauter, C, Shlyakhtenko, LS, Lyubchenko, YL, Guo, P & Chi, YI 2013, 'Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA', RNA, vol. 19, no. 9, pp. 1226-1237. https://doi.org/10.1261/rna.037077.112
Zhang, Hui ; Endrizzi, James A. ; Shu, Yi ; Haque, Farzin ; Sauter, Claude ; Shlyakhtenko, Lyudmila S. ; Lyubchenko, Yuri L ; Guo, Peixuan ; Chi, Young In. / Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA. In: RNA. 2013 ; Vol. 19, No. 9. pp. 1226-1237.
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abstract = "The bacteriophage phi29 DNA packaging motor, one of the strongest biological motors characterized to date, is geared by a packaging RNA (pRNA) ring. When assembled from three RNA fragments, its three-way junction (3WJ) motif is highly thermostable, is resistant to 8 M urea, and remains associated at extremely low concentrations in vitro and in vivo. To elucidate the structural basis for its unusual stability, we solved the crystal structure of this pRNA 3WJ motif at 3.05 {\AA}. The structure revealed two divalent metal ions that coordinate 4 nt of the RNA fragments. Single-molecule fluorescence resonance energy transfer (smFRET) analysis confirmed a structural change of 3WJ upon addition of Mg2+. The reported pRNA 3WJ conformation is different from a previously published construct that lacks the metal coordination sites. The phi29 DNA packaging motor contains a dodecameric connector at the vertex of the procapsid, with a central pore for DNA translocation. This portal connector serves as the foothold for pRNA binding to procapsid. Subsequent modeling of a connector/pRNA complex suggests that the pRNA of the phi29 DNA packaging motor exists as a hexameric complex serving as a sheath over the connector. The model of hexameric pRNA on the connector agrees with AFM images of the phi29 pRNA hexamer acquired in air and matches all distance parameters obtained from cross-linking, complementary modification, and chemical modification interference.",
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