The study of viral genome encapsidation has attracted increasing attention recently as it could reveal potential targets for anti-viral therapy and have relevance to other biochemical processes, such as molecular motion, bioengergetics, and macromolecular interactions. Extensive investigation on DNA packaging of the double-stranded DNA viruses documents certain common features in this step of the viral life cycle. Bacteriophage phi 29 encodes a small RNA (pRNA) that is indispensable for genomic DNA packaging. Recent work has revealed that several antisense oligonucleotides to pRNA completely block virion assembly in vitro, and that mutant pRNAs completely block virion assembly in vivo. The long term objective of this study is to determine the largely unknown role of the pRNA in phi 29 DNA packaging, to revela the basis of RNA/protein, RNA/DNA and RNA/RNA interactions, as well as to elucidate the mechanism of the RNA- involved, energy-driven DNA translocation into the procapsid. In this proposal, specific bases or gions of the pRNA will be labeled by the use of circularly permuted pRNAs and bipartite pRNAs. Intramolecular interactions of the aforementioned labeled pRNA as well as its interactions with other components involved in DNA packaging will be investigated by means of crosslinking. Intramolecular crosslinking will also be used to study conformational changes of the pRNA in the presence of various components of the DAN packaginh machine. The tertiary structure of the pRNA will be determined based on the previous solving of the pRNA secondary structure.