Functional analysis of fractionated Drosophila Kc cell tRNA gene transcription components

Abstract

We have previously described the partial purification of RNA polymerase III transcription components from Drosophila Kc cell extracts (Burke, D. J., Schaack, J., Sharp, S., and Söll, D. (1983) J. Biol. Chem. 258, 15224-15231). Here we report further physical and functional analysis of the two transcription-required fractions designated as factors B and C. Using stable complex formation-competition experiments with ordered addition of these transcription components, we have shown that factor C associates rapidly with the tRNA gene, but will not form a stable complex with the DNA in the absence of factor B. Reconstitution experiments of the individual Drosophila Kc cell factors with those from human HeLa cells demonstrated that the HeLa factor B, combined with Kc factor C, can form active transcription complexes. These now exhibit a property of the Drosophila system, a strong dependence on sequences in the 5'-flanking regions of tRNA genes. However, the Kc factor B (which has an apparent molecular weight of 260,000 as judged by gel filtration analysis) is not compatible with the HeLa factor C. These results, together with the isolation of a large complex containing all necessary transcription components, emphasize the importance of the interaction of these components for both stable complex formation and transcription initiation.