Analysis of RNA polymerase III transcription complexes by gel filtration

Abstract

We studied the in vitro assembly and stability of RNA polymerase III transcription complexes on the 5 S RNA and tRNAMet genes of Xenopus and the VA genes of adenovirus. Complete and partial assemblies were formed on these genes using transcription factor IIIA from Xenopus ovaries and factors IIIB and IIIC from HeLa cells. The complexes were purified away from unbound factors by filtration through Sepharose 4B columns and then assayed for transcription in the presence of Xenopus polymerase III. The 5 S gene complexes were also investigated using a postlabeling DNase I footprinting technique that we devised. The binding of factor IIIA to the 5 S gene facilitated the binding of factor IIIC; this subassembly was required for factor IIIB to bind. On the VA I and tRNA genes, factor IIIC alone bound and allowed IIIB to bind. RNA polymerase bound last to form a preinitiation complex, but it was less stably affixed than any of the factors. The complete factor complexes on the 5 S and VA I genes were strikingly stable to brief exposure to high salt concentrations, and the stability of the factor IIIB interaction was limiting. Two modes of IIIC binding were distinguished that differed in stability and specificity. Assembly of the complexes did not require ATP, and faithful transcription occurred when adenyl-5'-yl imidodiphosphate was substituted for ATP.