Identification of a region of the tobacco mosaic virus 126- and 183-kilodalton replication proteins which binds specifically to the viral 3'-terminal tRNA-like structure.
UV irradiation of a mixture of an isolated tobacco mosaic virus (TMV; tomato strain L [TMV-L]) RNA-dependent RNA polymerase complex and the TMV-L RNA 3'-terminal region (3'-TR) resulted in cross-linking of the TMV-L 126-kDa replication protein to the TMV-L 3'-TR. Using both Escherichia coli-expressed proteins corresponding to parts of the 126-kDa protein and mutants of the 3'-TR, the interacting sites were located to a 110-amino-acid region just downstream of the core methyltransferase domain in the protein and a region comprising the central core C and domain D2 in the 3'-TR. Mutation to alanine of a tyrosine residue at position 409 or a tyrosine residue at position 416 in the protein binding region abolished cross-linking to the 3'-TR, and corresponding mutations introduced into TMV-L RNA abolished its ability to replicate in tomato protoplasts, with no detectable production of either plus- or minus-strand RNA. The results are compatible with a model for initiation of TMV-L minus-strand RNA synthesis in which an internal region of the TMV-L 126-kDa protein first binds to the central core C and domain D2 region of the TMV-L 3'-TR and is then followed by binding of the 183-kDa protein to this complex and positioning of the catalytically active site of the polymerase domain close to the 3'-terminal CCCA initiation site.
Osman
T A M
TA
Department of Biological Sciences, Imperial College London, London SW7 2AZ, United Kingdom.
Buck
K W
KW
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D018551:Solanum lycopersicum; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011523:Protoplasts / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D014027:Tobacco Mosaic Virus / Q000502:physiology*; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D012367:RNA, Viral; D014764:Viral Proteins; D012343:RNA, Transfer