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 Mg 2+ . 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.

Diverse self-association properties within a family of phage packaging RNAs

“…The crystal structure of a tetrameric form of phi29 pRNA showed helices P A and P C to be coaxially stacked with helix P C emerging at an angle of ∼70° (Ding et al 2011). The crystal structure of the isolated phi29 3wj core showed similar helical arrangements, with some difference in the angle between P A and P C (Zhang et al 2013). Interestingly, when the phi29 pRNA was studied using solution methods such as single-molecule FRET and electron paramagnetic resonance, the resultant models show a coaxial stacking of P C and P D , with P A emerging at an angle, a conformation also suggested by computer modeling (Hoeprich and Guo 2002;Shu et al 2010;Zhang et al 2012).…”

“…In addition to the kissing interaction, it has been reported that the three-way junction within the phi29 pRNA has special properties including unique thermostability , or functionally important flexibility (Ding et al 2011). Recent crystal structures of an engineered phi29 pRNA (Ding et al 2011), and of an isolated phi29 pRNA three-way junction (lacking the kissing interactions) (Zhang et al 2013) provided additional details of the intermolecular and intramolecular interactions that are important within this specific pRNA species. The knowledge resulting from these diverse studies of the phi29 pRNA has led to its adaptation as a polyvalent delivery vector and it has been useful for various types of RNA engineering (Guo et al , 2006Li et al 2009;Wendell et al 2009;Shu et al 2011;Tarapore et al 2011;Ye et al 2012;Khisamutdinov et al 2014).…”

Thermodynamic stabilities of three-way junction nanomotifs in prohead RNA

“…A truncated phi29 construct with interlocking loops at 3.5 Å shows metal ions bound near the GU closing pair of the 3WJ in the native sequence (Ding et al 2011). A three-component model for the phi29 pRNA 3WJ motif at 3.05 Å was stabilized by two divalent metal ions binding between two helices (Zhang et al 2013). Comparison of the two metal-ion bound structures (Ding et al 2011;Zhang et al 2013) indicates a change in the A and D interhelical angle and helix distortion where the metal ions bind (Zhang et al 2013).…”

“…A three-component model for the phi29 pRNA 3WJ motif at 3.05 Å was stabilized by two divalent metal ions binding between two helices (Zhang et al 2013). Comparison of the two metal-ion bound structures (Ding et al 2011;Zhang et al 2013) indicates a change in the A and D interhelical angle and helix distortion where the metal ions bind (Zhang et al 2013). EPR methods determined another multibranch loop conformation in solution in a pRNA dimer in the presence of Mg 2+ (Zhang et al 2012).…”

“…In prohead RNA (pRNA), an essential component of the phi29-like bacteriophage DNA packaging motor (Guo et al 1987), the three-way junction (3WJ) is a multibranch loop that is a flexible, dynamic region in the RNA (Lescoute and Westhof 2006;Zhang et al 2012Zhang et al , 2013) that helps to correctly place helices in the spatial orientation necessary for packaging (Zhang et al 1997;Hoeprich and Guo 2002;Ding et al 2011;Hill et al 2016). In vitro, pRNA self-assembles, with some sequences capable of forming dimers, trimers, and higher-order multimers depending on the assembly conditions used.…”