Global Structure of a Three-Way Junction in a phi29 Packaging RNA Dimer determined using Site-Directed Spin Labeling

Abstract

The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/RNA complex, and its function requires an RNA component, called the packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA 3-way junction, which bridges binding sites for the motor ATPase and the procapsid. The studies were carried out in a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals (R5) were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 inter-R5 distances spanning the 3-way junction were measured using Double Electron-Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select approximately 3,600 viable 3-way junction models from a pool of 65 billion. The results reveal a similar conformation among all the viable models, with two of the helices (HT and HL) adopting an acute bend. This contrasts to a recently reported pRNA tetramer crystal structure, in which HT and HL stack onto each other linearly. The studies reveal versatility in pRNA conformations that may be beneficial to packaging motor function, and establish a new venue for mapping global structures of complex RNA molecules.