Minimal sequence and structural requirements of a subgenomic RNA promoter for turnip crinkle virus

Infection of plants or protoplasts with turnip crinkle Virus (TCV) results in the synthesis of the genomic RNA and two subgenomic (sg) RNAs of 1.7 kb and 1.45 kb, respectively. Both of the sgRNA promoters were characterized p reviously and their secondary structures predicted by computer analysis [J. Wang and A. E. Simon (1997). Virology 232, 174-186]. Secondary structure-s ensitive chemical and enzymatic probes have now been used to determine the structure of the promoter directing synthesis of the 1.45-kb sgRNA, namely the 1.45-kb sgRNA promoter, in solution. The newly obtained structure confo rms with the previously predicted hairpin structure except for the hairpin base: four CG base pairs and a CA bulge are present instead of an A bulge. Studies of deletions within the 96-nucleotide (nt) 1.45-kb sgRNA promoter d efined a minimal 30-nt core sequence as essential for promoter activity: a 21-nt hairpin and a 9-nt flanking single-stranded sequence. Mutational anal ysis in the stem section of the core promoter supported a role for the prim ary sequence and secondary structure in promoter activity. Sequence alterat ions in the flanking single-stranded region further suggest that the sequen ce CCCAUUA, encompassing the transcription start site, is required for effi cient transcription of the 1.45-kb sgRNA by the TCV RNA-dependent RNA polym erase in vivo. (C) 1999 Academic Press.