Mapping of the Tobacco mosaic virus movement protein and coat protein subgenomic RNA promoters in vivo

The Tobacco mosaic virus movement protein (MP) and coat protein (CP) are ex pressed from 3'-coterminal subgenomic RNAs (sgRNAs). The transcription star t site of the MP sgRNA, previously mapped to positions 4838 (Y. Watanabe, T . Meshi, and Y. Okada (1984), FEES Lett 173, 247-250) and 4828 (K. Lehto, G . L. Grantham, and W. O. Dawson (1990), Virology 174, 145-157) for the TMV OM and U1 strains, respectively, has been reexamined and mapped to position 4838 for strain U1. Sequences of the MP and CP sgRNA promoters were deline ated by deletion analysis. The boundaries for minimal and full MP sgRNA pro moter activity were localized between -35 and +10 and -95 and +40, respecti vely, relative to the transcription start site. The minimal CP sgRNA promot er was mapped between -69 and +12, whereas the boundaries of the fully acti ve promoter were between -157 and +54. Computer analysis predicted two stem -loop structures (SL1 and SL2) upstream of the MP sgRNA transcription start site. Deletion analysis and site-directed mutagenesis suggested that SL1 s econdary structure, bur not its sequence, was required for MP sgRNA promote r activity, whereas a 39-nt deletion removing most of the SL2 region increa sed MP sgRNA accumulation fourfold. Computer-predicted folding of the fully active CP sgRNA promoter revealed one long stem-loop structure. Deletion a nalysis suggested that the upper par? of this stem-loop, located upstream o f the transcription start site, was essential for transcription and that th e lower part of the stem had an enhancing role, (C) 2000 Academic Press.