The in vitro assembly of phage phi29 provides a system for developing methods to determine the stoichiometry of components in complex systems. Assembly from purified constituents generates 108 virions per ml, so this is an especially sensitive system for examining the effects of specific mutants. Two complementary methods have been used to determine the stoichiometry of an RNA oligomer that is required for DNA packaging of the phi29 capsid. In the first method empirical data are compared to theoretical curves generated by a binomial expression, assuming certain stoichiometries. In the second method, the dilution curves (log/log plots of component concentration versus yield of product) of components of known stoichiometry are compared to that of the unknown. The objective of the current proposal is to use these same methods to determine the stoichiometry of two additional constituents of phi29, gp16 and gp12. In the case of the former protein the analysis is complicated by the fact that gp16 acts catalytically in the DNA packaging step. This provides an opportunity to examine the influence of reusability on these two methods for determining stoichiometry. It is hoped that the types of analysis developed here will be used to study other complex biological systems.
