τ91, an Essential Subunit of Yeast Transcription Factor IIIC, Cooperates with τ138 in DNA Binding

ABSTRACT

Transcription factor IIIC (TFIIIC) (or τ) is a large multisubunit and multifunctional factor required for transcription of all class III genes in Saccharomyces cerevisiae. It is responsible for promoter recognition and TFIIIB assembly. We report here the cloning and characterization of TFC6, an essential gene encoding the 91-kDa polypeptide, τ91, present in affinity-purified TFIIIC. τ91 has a predicted molecular mass of 74 kDa. It harbors a central cluster of His and Cys residues and has basic and acidic amino acid regions, but it shows no specific similarity to known proteins or predicted open reading frames. The TFIIIC subunit status of τ91 was established by the following biochemical and genetic evidence. Antibodies to τ91 bound TFIIIC-DNA complexes in gel shift assays; in vivo, a B block-deficient U6 RNA gene (SNR6) harboring GAL4 binding sites was reactivated by fusing the GAL4 DNA binding domain to τ91; and a point mutation in TFC6 (τ91-E330K) was found to suppress the thermosensitive phenotype of a tfc3-G349Emutant affected in the B block binding subunit (τ138). The suppressor mutation alleviated the DNA binding and transcription defects of mutant TFIIIC in vitro. These results indicated that τ91 cooperates with τ138 for DNA binding. Recombinant τ91 by itself did not interact with a tRNA gene, although it showed a strong affinity for single-stranded DNA.

ACKNOWLEDGMENTS

We are grateful to Anny Ruet and Yveline Frobert for help in raising mouse polyclonal antibodies, to Janine Huet and Emmanuel Favry for recombinant TBP, recombinant TFIIIB70, and Pol III preparations, and to Martin Lanzendörfer for advice on recombinant protein purification and single-stranded DNA binding. We thank Michel Riva and Françoise Bouet for their help in peptide microsequence determination. We thank Christian Marck for pointing out to us the presence of τ131 and τ95 homologs in data banks and for helpful discussions, and we thank Carl Mann for improving the manuscript.

This work was supported by a grant from the European Union BIOTECH program (to A.S.). R.A. was supported by a long-term postdoctoral FEBS fellowship. N.M. and S.R. were supported by a fellowship from the French Ministére de l’Enseignement Supérieur et de la Recherche.