Virology 253, 327-336 (1999)

Minimal Sequence and Structural Requirements of A Subgenomic RNA Promoter for Turnip Crinkle Virus

Jianlong Wang, Clifford D. Carpenter and Anne E. Simon

Department of Biochemistry and Molecular Biology and Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003

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- and 1.45-kb, respectively. Both of the sgRNA promoters were characterized previously and their secondary structures predicted by computer analysis (J. Wang and A. E. Simon, (1997). Virology 232, 174-186). Secondary structure-sensitive 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 conforms 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 defined a minimal 30-nt core sequence as essential for promoter activity: a 21-nt hairpin and a 9-nt flanking single-stranded sequence. Mutational analysis in the stem section of the core promoter supported a role for the primary sequence and secondary structure in promoter activity. Sequence alterations in the flanking single-stranded region further suggests that the sequence CCCAUUA, encompassing the transcription start site, is required for efficient transcription of the 1.45-kb sgRNA by the TCV RNA-dependent RNA polymerase in vivo.