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

doi:10.1074/jbc.M600101200

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Papers In Press, published online ahead of print March 3, 2006
J. Biol. Chem, 10.1074/jbc.M600101200
Submitted on January 5, 2006
Revised on February 23, 2006
Accepted on March 3, 2006

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

Cécile Ducrot, Olivier Lefebvre, Emilie Landrieux, Josée Guirouilh-Barbat, André Sentenac, and Joel Acker

DSV/DBJC/SBGM, CEA Saclay, Gif sur yvette 91191

Corresponding Author: joel.acker{at}cea.fr

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, tA and tB, using the baculovirus expression system. The B-block binding module, rtB, was reconstituted with rt138, rt91 and rt60 subunits. rt131, rt95 and rt55 formed also a stable complex, rtA, that displayed non-specific 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.

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