Mapping of the Tobacco Mosaic Virus Movement Protein and Coat Protein Subgenomic RNA Promoters in Vivo

Abstract

The Tobacco mosaic virus movement protein (MP) and coat protein (CP) are expressed from 3′-coterminal subgenomic RNAs (sgRNAs). The transcription start site of the MP sgRNA, previously mapped to positions 4838 (Y. Watanabe, T. Meshi, and Y. Okada (1984), FEBS 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 delineated by deletion analysis. The boundaries for minimal and full MP sgRNA promoter activity were localized between −35 and +10 and −95 and +40, respectively, relative to the transcription start site. The minimal CP sgRNA promoter was mapped between −69 and +12, whereas the boundaries of the fully active 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 secondary structure, but not its sequence, was required for MP sgRNA promoter activity, whereas a 39-nt deletion removing most of the SL2 region increased MP sgRNA accumulation fourfold. Computer-predicted folding of the fully active CP sgRNA promoter revealed one long stem–loop structure. Deletion analysis suggested that the upper part of this stem–loop, located upstream of the transcription start site, was essential for transcription and that the lower part of the stem had an enhancing role.