Multiple factors are required for the accurate transcription of purified genes by RNA polymerase III.

Cell-free extracts (S-100) prepared from cultured mammalian KB cells have previously been shown to direct accurate and selective transcription of class III genes by RNA polymerase III. We have fractionated the KB S-100 and have found that multiple components are essential for the accurate transcription of these genes. After the S-100 has been separated into four different protein fractions by chromatography on phosphocellulose, two fractions are required, in addition to RNA polymerase III, for active and selective transcription of the virus-associated RNAI gene of adenovirus 2 and a tRNA gene; a third fraction is required, along with these components, for the reconstitution of 5 S RNA gene transcription. At least two of these components are distinct from the four factors required for accurate initiation of transcription by RNA polymerase II (Matsui, T., Segall, J., Weil, P. A., and Roeder, R. G. (1980) J. Biol. Chem. 255, 11992-11996).