FIG. 1.

Purification of RAF-2. (A) General scheme for purification of RAF-2. The details of column chromatography and the biochemical complementation assay system are described in Materials and Methods. (B) Gel filtration profile of RAF-2 activity. RNA synthesis was carried out in the absence (−) or presence of 0.34 M KCl eluate from the second Mono Q column (input, 1.5 μl) and aliquots of fractions separated through a gel filtration column (lanes 1 to 11, 1.5 μl of each). Arrowheads indicate the elution positions of molecular weight marker proteins (Bio-Rad). RAF-2 activity is observed in fractions corresponding to a molecular mass of 60 to 80 kDa (lanes 7 and 8). (C) Proteins present in the RAF-2 fraction. Five microliters of each fraction was loaded onto a 10% polyacrylamide gel containing 1% SDS. Electrophoresis was carried out, and the polypeptides were visualized by Coomassie brilliant blue staining. The RAF-2 fraction (lanes 7 and 8) contains 48-kDa and 36-kDa polypeptides as the major components (arrowheads). M, markers. (D) Stimulatory activity of RAF-2 in in vitro RNA synthesis systems containing different enzyme sources. In vitro RNA synthesis was carried out with mnRNP (lane 1) and vRNP (lanes 2 and 3). The level of RNA synthesis was determined by scanning the autoradiogram with the NIH Image analysis program. Ratios of the amounts of RNA products synthesized from the 53-mer model vRNA (lanes 1 and 2) or endogenous vRNAs (lane 3) in the presence of purified RAF-2 to those in the absence of purified RAF-2 are indicated as the average of three independent experiments. To determine the level of RNA synthesis from endogenous vRNAs, RNA products corresponding to all eight segments were included. (E) Stimulatory activity of RAF-2 in in vitro RNA synthesis systems with different RNA templates. In vitro RNA synthesis was carried out in the absence (lanes 1 and 5) or presence of 0.1 (lanes 2 and 6), 0.2 (lanes 3 and 7), and 0.4 (lanes 4 and 8) μl of purified RAF-2, using equimolar amounts of 53-mer (lanes 1 to 4) and 172-mer (lanes 5 to 8) model vRNA templates. The 172-mer RNA containing the same 5′- and 3′-terminal sequences as the 53-mer RNA was prepared as described elsewhere (54). RNA synthesis activity is shown as the ratio (the average of two independent experiments) of the amount of RNA products synthesized in the presence of RAF-2 to the amount synthesized in the absence of RAF-2.