The role of ATP in the biogenesis of vaccinia virus mRNA in vitro

Abstract

During in vitro RNA synthesis vaccinia virus rapidly depletes ATP but not GTP, CTP, or UTP. This reduction in ATP concentration results in a decreased rate of RNA synthesis. The RNA transcripts made when ATP becomes rate-limiting are large in size, 20 to 30 S, compared with the 8 to 12 S RNA transcripts synthesized under high ATP concentration. Furthermore, these large RNA transcripts are not significantly polyadenylated and remain virion-associated. When one supplements the RNA polymerase reaction with additional ATP the following observations are made: (a) The rate of RNA synthesis remains constant; (b) the RNA synthesized is of the appropriate monocistronic size, 8 to 12 S; (c) the RNA is polyadenylated efficiently; and (d) the RNA is released from the virus. Some of these effects are mimicked by dATP, but neither the αβ- nor the β,γ-phosphonic acid analogs of ATP will replace ATP in the supplementation scheme. Selection of appropriate ATP concentrations in the in vitro RNA polymerase reaction allows exclusive synthesis of high-molecular-weight RNA.