Isolation from tobacco mosaic virus-infected tobacco of a solubilized template-specific RNA-dependent RNA polymerase containing a 126K/183K protein heterodimer

The complete nucleotide sequence was determined for the putative RNA polyme rase (183K protein) gene of tobacco mosaic virus (TMV) OM strain, which dif fered from the related strain, vulgare, by 51 positions in its nucleotide s equence and 6 residues in its amino acid sequence. Three segments of this 1 83K protein, each containing the sequence motif of methyltransferase (M), h elicase (H), or RNA-dependent RNA polymerase (P), were expressed in Escheri chia call as fusion proteins with hexahistidine tags, and domain-specific a ntibodies were raised against purified His-tagged M and P polypeptides. By immunoaffinity purification, a template-specific RNA-dependent RNA polymera se containing a heterodimer of the full-length 183K and 126K tan amino-term inal-proximal portion of the 183K protein) viral proteins was isolated. We propose that the TMV RNA polymerase for minus-strand RNA synthesis is compo sed of one molecule each of the 183- and 126-kDa proteins, possibly togethe r with two or more host proteins.