RNA PROMOTERS LOCATED ON (-)-STRANDS OF A SUBVIRAL RNA ASSOCIATED WITH TURNIP CRINKLE VIRUS

Satellite (sat-) RNA C, one of the nonessential subviral RNAs of turni p crinkle virus (TCV), is dependent on the TCV-encoded RNA-dependent R NA polymerase (RdRp) for its replication. Earlier work showed that a s tem-loop structure at the 3' end of (+)-strand sat-RNA C is required f or synthesis of (-)-strands in vitro using a partially purified, templ ate-specific TCV RdRp (Song C, Simon AE, 1995, J Mol Biol 254:6-14). C is-sequences on (-)-strands of sat-RNA C that can serve as separate pr omoters in vitro have now been defined. Two promoter sequences are loc ated on (-)-strand sat-RNA C, one comprising 11 bases located near the 3' end, and the other consisting of 14 bases located 41 bases from th e 5' end. Both promoter sequences contain multiple consecutive C resid ues followed by multiple consecutive purines and have no obvious secon dary structure, suggesting that, along with hairpin structures, specif ic primary sequences can be recognized by the TCV RdRp. The 3'-proxima l promoter sequence directed synthesis from the 3' terminus using (-)- strand templates with the natural sat-RNA 3' end (AUCCC-3'). When plas mid-derived bases were present at the 3' ends of the templates, both p romoter sequences could direct the RdRp to initiate transcription inte rnally at the multiple consecutive C residues within the promoters. Th is result suggests that multiple consecutive C residues are important for transcription initiation and that natural 3'-end sequences, when l ocated at 3' termini, help the RdRp to initiate at the 3' end of the m olecule.