Book of Abstracts: Albany 2007
June 19-23 2007
Mapping the Global Structure of phi29 Packaging RNA Using Site-directed Spin Labelinga
The phi29 packaging RNA (pRNA) is an essential component in the strongest bio-molecular motor -- the phi29 bacteriophage DNA packaging motor. Utilizing Mg2+-dependent inter-molecular base pairing, multiple copies of pRNA form a ring-shaped complex that is indispensable in motor ATPase activity. To expand the limited knowledge on pRNA structure, we are utilizing the site-directed spin labeling (SDSL) technique to map the global structure of a pRNA closed dimer, which is a functional pRNA species and partially mimics pRNA/pRNA interaction in the motor. SDSL obtained local structural and dynamic information via electron paramagnetic resonance spectroscopy of a stable nitroxide radical attached site-specifically to a macromolecule. We have developed SDSL methods for accurately measuring distances ranging from 5-50 Å in arbitrary DNA or RNA sequences. These methods were successfully applied to measure multiple distances ranging from 30-45 Å in pRNA closed dimers. These are the first successful measurements of distance > 20 Å in any pRNA species. The distance constraints will be used to determine the relative positioning of the individual pRNA domains; thus, yielding the global structure of the pRNA closed dimer. These studies are the first application of SDSL to an RNA with unknown structure. Information obtained on pRNA will aid in testing our proposed ?rigid body motion? model, where ATP dependent movements within a pRNA monomer sequentially activate each monomer and drive DNA packaging. This will advance our understanding on the packaging motor function, and enable new pRNA based nano-technology developments.
Peter Z. Qin
Department of Chemistry and Department of Biological Sciences