Moieties in an RNA promoter specifically recognized by a viral RNA-dependent RNA polymerase

RNAs 33 nucleotides in length can direct accurate initiation of subgenomic RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase (RdRp), provided that the native sequences are maintained at five positions: -17, -14, -13, -11, and the +1 initiation site. The functional groups in the bases of these essential nucleotides required to interact with RdRp were examined by using chemically synthesized RNAs containing base analogs at each of the five positions. Analysis using a template competition assay revealed that the mode of recognition for the initiation nucleotide (+1) is distinct from that of the other essential nucleotides in the promoter. Competition experiments also determined that three template nucleotides are sufficient for stable interaction with RdRp. These results identify base moieties in the brome mosaic virus subgenomic promoter required for efficient RNA synthesis and support the hypothesis that the recognition of a RNA promoter by a viral RdRp is analogous to the recognition of DNA promoters by DNA-dependent RNA polymerases.