Replication of viral RNA, 8. Studies on the enzymatic mechanism of replication of MS2 RNA.

Abstract

The experiments reported in this paper show that, under certain conditions, the product of RNA synthetase is largely RNase-sensitive and therefore not in a hydrogen-bonded double helix. Following treatment with protein-denaturing agents (phenol, sodium dodecylsulfate), a large proportion of this material is rendered resistant to RNase with extraordinary ease, presumably by being converted into a double-helical form. The evidence so far available suggests that in vivo virus-specific double-stranded RNA (replicative form) provides the template for the formation of parental-type "plus" RNA strands. Synthesis proceeds by an asymmetric replication mechanism of a semiconservative nature, in which the new "plus" strand displaces the old one from the duplex. It would appear that under in vitro conditions the enzyme holds the growing "plus" strand in proximity-but without hydrogen bonding-to the "minus" strand of the template. This complex may, in vitro, be prevented from spontaneoulsy giving rise to a duplex by attachment of proteins or ribosomes to the single strands. Removal of protein would allow hydrogen bonding to be established. In pulse-labeling experiments in vivo, substantial amounts of RNase-resistant RNA are found even prior to phenol treatment. While these results might be interpreted to mean that the formation of the RNase-sensitive complex, described above, could be a peculiarity of the in vitro reaction, they could also be explained by assuming that the complex has but a transient existence in vivo.