Migration or translocation of macromolecules, such as proteins or nucleic acids, through barriers or cell membrane is a common process in biological system. One of the most complex and intricate translocation processes is viral DNA encapsidation. All linear dsDNA viruses, including hepesviruses, poxviruses, adenoviruses, and the ds-DNA bacteriophages package their genomic DNA into a preformed protein shell (procapsid). ATP is consumed to provide energy to condense the lengthy genomic DNA into the limited space inside procapsids with remarkable velocity. Phage phi29 requires a viral-encoded 120-base RNA (pRNA) to package its genomic DNA. This pRNA contains two single-stranded loops that are responsible for inter-RNA interactions. Through this loop/loop interaction, six pRNAs form a hexameric ring to gear the DNA packaging machine. We hypothesize that the pRNA possesses at least two functional conformations. Alternating between contraction and relaxation, driven by ATP hydrolysis, of each member of the pRNA hexagon could help rotate the DNA transportation machinery. Our long term objective is to elucidate how this DNA transportation motor works, thus providing fundamental information concerning bioenergetics as well as the mechanism of the interactions among protein, DNA and RNA. The short term objective of this proposal is to investigate the conditions for the formation of pRNA dimers or hexamers, and to construct hexagonal arrays composed of hexameric pRNA complex for future structural analysis.
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