Migration or translocation of proteins or nucleic acids through barriers or cell membranes is a common process in biological systems. One of the most complex and intricate translocation processes is viral genomic DNA packaging. A strikingly common feature in the maturation of linear dsDNA viruses, including herpes viruses, adenoviruses, and the ds-DNA bacteriophages, is that the lengthy genome is translocated with remarkable velocity into a limited space within a preformed protein shell and packaged into near-crystalline density. A DNA-packaging motor accomplishes this energetically unfavorable motion reaction. The bacterial virus phi29 DNA packaging motor contains six pRNA molecules, which bind ATP and form a hexamer as a vital part of the motor. This pRNA contains two function domains, one domain is for binding to the protein component of the motor, and the other is the DNA-packaging domain, but the detailed structure and function of this domain is unknown. We hypothesize that the alternation between contraction and relaxation, driven by ATP hydrolysis, of each member of the hexameric RNA ring could rotate the DNA transportation machinery. Our long-term objective is to elucidate the biochemical and biophysical mechanism of this RNA-containing motor, thus providing fundamental information concerning antiviral therapy, bioenergy conversion, macromolecular interactions, nanomotors as parts for nanodevices, and the general mechanism(s) of biomotors. For instance, the motor components of cytomegalovirus have been used as targets for antiviral drug design. The short-term objective of this renewed grant is to continue the study on the structure and function of the hexamer pRNA, focusing on the structure and function of the DNA packaging domain. The stoichiometry of pRNA on the motor before, during and after DNA packaging will be confirmed. ATP-binding and possible ATPase activity of pRNA will be further investigated by crosslinking and ATPase assays under various conditions. Photoaffinity crosslinking will be used to determine the target, if any, the DNA-packaging domain binds or is adjacent to. The specific pRNA/protein interactions will be probed to refine our understanding of the 3D structure of the motor complex. Probing for such precise interactions involves the detection of both the specific nucleotides of pRNA that bind to the connector and the specific amino acids of the connector protein that contacts the pRNA. Fluorescent beads or nanotubes will be used to label the components to allow direct observation of the direction of motion.