Lysis in cells infected with the ssRNA phage Qbeta is effected by the A2 protein. It was previously shown that a single copy of A2 assembled on the surface of the Qbeta virion inhibited the activity of MurA, which catalyzes the first committed step of murein biosynthesis. This led to a model for lysis timing in which A2 is not active as a MurA inhibitor until assembled into virion particles. Here we report that MurA inactivates purified Qbeta particles. Moreover, over-expression of MurA does not inactivate particles during the Qbeta infection cycle; thus, casting doubt on the notion that completed virions could be the lytic agent in vivo and also that the MurA-virion interaction does not occur in the infected cell. Furthermore, RNA released from particles was found to protect virions from inactivation by MurA in vitro, suggesting that Qbeta RNA might serve as the protective element during the infection cycle. Comparison of A2 accumulation between Qbeta and Qbeta^por mutants, which are Qbeta A2 mutants with a shorter infection cycle and reduced burst size, reveals that a delicate balance between assembled and unassembled A2 levels regulates lysis timing. A new model is proposed in which "free", unassembled A2 inhibits MurA. From in vitro binding studies and genetic analyses it was determined that A2 binds MurA in a closed conformation with UDP-N-acetylglucosamine bound.