RNA promoters located on (-)-strands of a subviral RNA associated with turnip crinkle virus

Abstract

Satellite (sat-) RNA C, one of the nonessential subviral RNAs of turnip crinkle virus (TCV), is dependent on the TCV-encoded RNA-dependent RNA polymerase (RdRp) for its replication. Earlier work showed that a stem-loop structure at the 3' end of (+)-strand sat-RNA C is required for synthesis of (-)-strands in vitro using a partially purified, template-specific TCV RdRp (Song C, Simon AE, 1995, J Mol Biol 254:6-14). Cis-sequences on (-)-strands of sat-RNA C that can serve as separate promoters in vitro have now been defined. Two promoter sequences are located 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 the 5' end. Both promoter sequences contain multiple consecutive C residues followed by multiple consecutive purines and have no obvious secondary structure, suggesting that, along with hairpin structures, specific primary sequences can be recognized by the TCV RdRp. The 3'-proximal promoter sequence directed synthesis from the 3' terminus using (-)-strand templates with the natural sat-RNA 3' end (AUCCC-3'). When plasmid-derived bases were present at the 3' ends of the templates, both promoter sequences could direct the RdRp to initiate transcription internally at the multiple consecutive C residues within the promoters. This result suggests that multiple consecutive C residues are important for transcription initiation and that natural 3'-end sequences, when located at 3' termini, help the RdRp to initiate at the 3' end of the molecule.