Mapping the Global Conformation of the Phi29 Packaging RNA Dimer Using Deer Distance Constraints

Abstract

The insertion 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-shape protein/RNA complex. The RNA component, called the packaging RNA (pRNA), is indispensable for motor function, and may play an essential role in motor ATPase activity. Current structural information on pRNA is limited, which hinders our effort on understanding motor function. Here, we use site-directed spin labeling and pulse EPR spectroscopy to map the global structure of a pRNA dimer that has been shown to be a functional intermediate in assembling the ring-shaped pRNA complex in the packaging motor. In our studies, nitroxide pairs were attached to specific sites of a truncated monomeric pRNA construct, the labeled monomers were then assembled into dimers in the presence of Mg2+, and inter-nitroxide distances were measured using DEER (Double Electron-Electron Resonance) spectroscopy. In parallel, an unbiased pool of models that contains variable pRNA conformations was generated, which treats pRNA as a 3-way junction construct, and a set of corresponding inter-nitroxide distances was predicted for each model. Intra-molecular DEER distances were used to obtain the monomeric structures of pRNA in dimer, which are then used to build the structural pool for pRNA dimers. A very small number of models were selected. We expect that this work will provide much-needed structural information regarding pRNA, as well as establishing a new methodology for analyzing global conformations in complex RNAs.