Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors

Abstract

Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, τA and τB, using the baculovirus expression system. The B block-binding module, rτB, was reconstituted with rτ138, rτ91, and rτ60 subunits. rτ131, rτ95, and rτ55 formed also a stable complex, rτA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B″ fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.