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J. Biol. Chem., Vol. 281, Issue 17, 11685-11692, April 28, 2006

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Reconstitution of the Yeast RNA Polymerase III Transcription System with All Recombinant Factors^*

From the Service de Biochimie et de Génétique Moléculaire, Bâtiment 144, CEA/Saclay, F-91191 Gif-sur-Yvette Cedex, France

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, A and B, using the baculovirus expression system. The B block-binding module, rB, was reconstituted with r138, r91, and r60 subunits. r131, r95, and r55 formed also a stable complex, rA, 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.

Received for publication, January 5, 2006 , and in revised form, February 23, 2006.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: CMU/Département de Microbiologie et de Médecine Moléculaire,Genève, Suisse.

² Present address: Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institute of Health, Bethesda, MD 20892-4255.

³ To whom correspondence should be addressed. Tel.: 33-1-69083796; Fax: 33-1-69084712; E-mail: joel.acker{at}cea.fr.

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