FIG. 3.

Model mini-vRNA template-directed RNA synthesis in vitro by TMV RNA polymerase. The model mini-vRNA template of 249 nucleotides, prepared by transcribing pTMV3′(+)-T7 template by T7 RNA polymerase, corresponds to 3′-terminal noncoding positive-strand RNA between positions 6147 and 6395 (the 3′ terminus). The immunoaffinity-purified RNA polymerase was added to the reaction mixture of the standard in vitro transcription assay, including the model mini-vRNA template and [α-³²P]UTP as a labeled substrate. The enzymes used were as follows: lanes 1 and 2, S100 fractions from healthy (H) and TMV-infected (I) tobacco; lane 3, no added enzyme; lanes 4 and 5, the RNA polymerase fractions isolated with anti-M antibodies after a washing with 30 bed volumes of the Tris-Triton-NaCl buffer; and lanes 6 and 7, the RNA polymerase fractions isolated with anti-P antibodies after a washing with a 30 bed volumes of the Tris-Triton-NaCl buffer. RNA products were analyzed by urea–6% PAGE, and the gel was visualized with a BAS-2000 image analyzer (Fuji). The positions of single-stranded RNA molecular-size markers are shown on the left.