The 174-base bacteriophage phi29-encoded prohead RNA (pRNA) is essential in the packaging of the 19 kilobase pair DNA-gene product 3 complex (DNA-gp3) into the viral precursor capsid or prohead. This finding may have general significance for assembly of viruses at this level of structural complexity. The ultimate goal is to determine the mechanism by which pRNA constitutes the phi29 DNA packaging machine and enjoins with proteins to catalyze DNA-gp3 translocation. The goal of the current project is to determine the higher order structure of pRNA as it constitutes the active center of the DNA packaging machine and to determine its geometry relative to the capsid portal protein gpl0 and the ATP-binding protein gpl6. The phi29 pRNA gene will be mutagenized by site-directed and random mutagenesis and the pRNA mutants identified and sequenced. Suppressors of specific mutants will be isolated and sequenced to identify pseudoknots and long range interactions. The mutant RNAs will be tested for interactions with proheads in DNA-gp3 packaging and for ATPase activity when complexed to gpl6. Photoactivatable azido-ATP will be used to identify the ATPase active center(s) of the protein and/or pRNA constituents of the DNA-gp3 packaging machine. The folding and higher order structure of pRNA alone and pRNA complexed to the prohead and/or gp 16 will be investigated by cleavage with Fe(II)-EDTA and Rh(phen)2phi3+ and bases derivatized with DTPA-FE(II) will be incorporated at selected sites on pRNA to target cleavage. UV cross-links will be sought within pRNA and pRNA-protein complexes under conditions of catalysis as a probe of three-dimensional structure. When tertiary constraints are obtained, a symbolic and numerical computation method will be used for three-dimensional modeling of pRNA. Attempts will be made to crystallize pRNA and to cocrystallize pRNA and gpl6 for x-ray diffraction studies. Finally, pRNA will be localized on proheads, and the step at which it leaves the nascent viral particle will be investigated. The mass, stoichiometry and morphology of pRNA-gp 16 and pRNA-connector complexes will be determined.
