title: Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA.
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.
authors.affiliation: Nanobiotechnology Center, Markey Cancer Center and Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky 40536, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.forename: James A
authors.initials: JA
authors.lastname: Endrizzi
authors.forename: Yi
authors.initials: Y
authors.lastname: Shu
authors.forename: Farzin
authors.initials: F
authors.lastname: Haque
authors.forename: Claude
authors.initials: C
authors.lastname: Sauter
authors.forename: Lyudmila S
authors.initials: LS
authors.lastname: Shlyakhtenko
authors.forename: Yuri
authors.initials: Y
authors.lastname: Lyubchenko
authors.forename: Peixuan
authors.initials: P
authors.lastname: Guo
authors.forename: Young-In
authors.initials: YI
authors.lastname: Chi
mesh_terms: D017103:Bacillus Phages / Q000235:genetics*; D001665:Binding Sites; D036022:Capsid Proteins / Q000737:chemistry; D002413:Cations, Divalent; D018360:Crystallography, X-Ray; D042003:DNA Packaging; D004279:DNA, Viral / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D009690:Nucleic Acid Conformation; D020871:RNA Stability; D012367:RNA, Viral / Q000737:chemistry*; D019065:Virus Assembly
chemical_list: D036022:Capsid Proteins; D002413:Cations, Divalent; D004279:DNA, Viral; D012367:RNA, Viral; D008274:Magnesium
keywords: RNA crystal; RNA nanotechnology; RNA therapeutics; metal ions; three-way junction