RNA Synthesis by the Brome Mosaic Virus RNA-Dependent RNA Polymerase: Transition from Initiation to Elongation

Abstract

The initiation and elongation phases of (−)-strand RNA synthesisin vitroby the brome mosaic virus RNA-dependent RNA polymerase (RdRp) are differentially sensitive to inhibitors. In an attempt to characterize further the transition RdRp makes from initiation to elongation, we determined the conditions needed to pause the ternary complex and complete only one round of RNA synthesis. During the transition we were able to discern step-wise increases in the affinity of RdRp for RNA by measuring sensitivity to heparin and competition for RdRp by an alternative template. Three distinct stability levels of RdRp–template interactions were found. The first stable RdRp–RNA complex was observed when RdRp bound to the RNA template. A further increase occurred when RdRp synthesized the first phosphodiester bond. A final increase occurred upon formation of between 3 and 13 phosphodiester bonds. After this last transition, RdRp appeared to be tightly committed to the template RNA. These results are analogous to the mechanism of action of DNA-dependent RNA polymerases and are relevant to protein–RNA interaction and template switching by an RdRp.