Genetic interactions within TFIIIC, the promoter-binding factor of yeast RNA polymerase III
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O R I GI N A L P A P E R
S. Rozenfeld á P. Thuriaux
Genetic interactions within TFIIIC, the promoter-binding factor of yeast RNA polymerase III
Received: 8 January 2001 / Accepted: 2 February 2001 / Published online: 30 March 2001 Ó Springer-Verlag 2001
Abstract TFIIIC is a heteromultimeric protein, made of six distinct subunits in Saccharomyces cerevisiae, that binds to RNA polymerase III promoters and triggers the assembly of the transcription complex. The largest yeast subunit tau138, encoded by TFC3, binds to the B-box promoter element. This binding is defective in the temperature-sensitive mutant tfc3-G349E; the mutation responsible is located in one of two conserved motifs shared with the B-binding component of human TFIIIC. Rare dominant gain-of-function mutations that restore growth at high temperature were obtained following ultraviolet mutagenesis of tfc3-G349E. All of them resulted from single amino acid substitutions that alter the structure of TFIIIC. Three were due to reversion or intragenic suppression (TFC3-K754E and TFC3-L804H) events. Three were identical isolates of TFC6-E330K, a previously described mutation of the tau91 subunit. The remaining suppressors mapped in TFC4, and resulted in amino acid replacements in the second largest subunit of TFIIIC (tau131). With the exception TFC4-E711K, these aect positions that are invariant between the S. cerevisiae and Homo sapiens proteins, and are localised in conserved tetratricopeptide motifs. These ®ndings demonstrate a close functional interaction between the two largest subunits of TFIIIC and underscore the importance of the tetratricopeptide motif of tau131. Keywords Yeast á Transfer RNAs á Tetratricopeptide á TFIIIB á Transcription
Communicated by E. Di Mauro S. Rozenfeld á P. Thuriaux (&) Service de Biochimie et GeÂneÂtique MoleÂculaire, CEA/Saclay, Bat. 142, CEA/Saclay, 91191 Gif-sur-Yvette Cedex, France E-mail: [email protected] Tel.: +33-1-69083586 Fax: +33-1-69084712
Introduction In eukaryotes, the synthesis of 5S rRNA, tRNAs and of several small untranslated RNAs depends on a large multiprotein complex organised around RNA polymerase III (Pol III). This complex has been well characterised in Saccharomyces cerevisiae, where the polymerase, its promoter-binding factor TFIIIC and the initiation factor TFIIIB are necessary and sucient for faithful transcription in vitro, producing complete transcripts that are identical to the primary transcripts found in vivo. The corresponding twenty-seven polypeptides have all been biochemically and genetically identi®ed, and are essential for growth (CheÂdin et al. 1998; Kumar et al. 1998). One of the most striking features of the Pol III complex is the very large size of its promoter-binding component. Yeast TFIIIC is almost as large (512 kDa) as the enzyme itself (617 kDa), and has six dierent subunits (Gabrielsen et al. 1989; Conesa et al. 1993; Huet et al. 1996). This undoubtedly re¯ects a multiplicity of biological roles. Indeed, TFIIIC recognises promoters (Camier et al. 1990), re
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