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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 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 three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on 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 were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 internitroxide distances spanning the three-way junction were measured using Double Electron-Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (H(T) and H(L)) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which H(T) and H(L) stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial.

This protocol describes the procedures for measuring nanometer distances in nucleic acids using a nitroxide probe that can be attached to any nucleotide within a given sequence. Two nitroxides are attached to phosphorothioates that are chemically substituted at specific sites of DNA or RNA. Inter-nitroxide distances are measured using a four-pulse double electron-electron resonance technique, and the measured distances are correlated to the parent structures using a Web-accessible computer program. Four to five days are needed for sample labeling, purification and distance measurement. The procedures described herein provide a method for probing global structures and studying conformational changes of nucleic acids and protein/nucleic acid complexes.

To better understand the mechanism of actin filament (F-actin) bundling by polyamines, we have measured the onset of bundling as a function of polyamine concentration. Samples were centrifuged at low speeds to separate bundles from unbundled actin, and the relative amounts of actin in the pellet and supernatant were determined via gel electrophoresis, yielding a description of the bundling transition as a function of actin and polyamine concentrations. These experiments were carried out for two different polyamines, spermine (tetravalent) and spermidine (trivalent). We found that the threshold concentration of polyamine needed to bundle actin is independent of both actin concentration and Mg2+ concentration over a wide range in Mg2+ concentration. We also find that spermine in F-actin bundles is essentially invisible in solution-phase proton NMR, suggesting that it is bound so tightly to F-actin that it is immobilized.