A minimal RNA promoter for minus-strand RNA synthesis by the brome mosaic virus polymerase complex

The approximately 150 nt tRNA-like structure present at the 3' end of each of the brome mosaic virus (BMV) genomic RNAs is sufficient to direct minus- strand RNA synthesis. RNAs containing mutations in the tRNA-like structure that decrease minus-strand synthesis were tested for their ability to inter act with RdRp (RNA-dependent RNA polymerase) using a template competition a ssay. Mutations that are predicted to disrupt the pseudoknot and stem B1 do not affect the ability of the tRNA-like structure to interact with RdRp. S imilarly, the +1 and +2 nucleotides are not required for stable template-Rd Rp interaction. Mutations in the bulge and hairpin loops of stem C decrease d the ability of the tRNA-like structure to interact with RdRp Furthermore, in the absence of the rest of the BMV tRNA, stem C is able to interact wit h RdRp. The addition of an accessible initiation sequence containing ACCA3' to stem C created an RNA capable of directing RNA synthesis. Synthesis fro m this minimal minus-strand template is dependent on sequences in the hairp in and bulged loops. (C) 1999 Academic Press.