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In previous studies, we have shown that the PCFI-I mutation of Saccharomyces cerevisiae suppresses the negative effect of a tRNA gene A block promoter mutation in vivo and increases the transcription of a variety of RNA polymerase III genes in vitro. Here, we report that PCF1 encodes the second largest subunit of transcription factor IIIC (TFIIIC) and that the PCFI-1 mutation causes an amino acid substitution in a novel protein structural motif, a tetratricopeptide repeat, in this subunit. polypeptide is the only TFIIIC subunit that can be photocross-linked to the region footprinted by TFIIIB. This subunit is a likely candidate to mediate the recruitment of TFIIIB. Similarly, the 95-and 55-kDa subunits may interact with the A block, and the 138-kDa subunit may interact with the B block. To help resolve the roles of individual TFIIIC subunits in transcription complex assembly, a major effort to clone these genes has been under way. So far, this has been achieved for the 95-, 131-, and 138-kDa polypeptides (23,26,29,34).Studies with yeast and human cells indicate that TFIIIB is a multisubunit factor (20,24,35,36). Detailed characterization of the yeast factor suggests that it comprises three components: the TATA-binding protein (TBP), a 70-kDa TFIIB-like polypeptide (TFIIIB70), and a 90-kDa polypeptide (TFIIIB90). These three polypeptides are stably associated under most conditions and copurify as a complex with TFIIIB activity over numerous columns. However, by using strong cation exchangers, specifically MonoS, TFIIIB activity has been separated into two fractions, designated TFIIIB ' and TFIIIB" (18). By photocross-linking, these two fractions were found to contain the TFIIIB70 and TFIIIBgo polypeptides, respectively, which had been identified previously in less pure TFIIIB fractions with this technique (3). Following the demonstration of a universal role for TBP in eukaryotic transcription (7, 32), TBP was identified as a component of TFIIIB and traced by Western blotting (immunoblotting) to the TFIIIB' fraction (20). This result together with the cloning of the gene for TFIIIB70 (5,6,25) has permitted the demonstration that these proteins are the only TFIIIB components in the TFIIIB' fraction. The two proteins expressed in bacteria can replace the TFIIIB' fraction in a reconstituted transcription system (20). The TFIIIB" fraction has not been purified to homogeneity. However, TFIIIB90 may be the only TFIIIB subunit in this fraction, since TFIIIB" transcription activity can be provided by sodium dodecyl sulfate (SDS) gel-eluted and renatured proteins in the 90-kDa size range (20

An important step in the assembly of RNA polymerase (pol) III transcription complexes on tRNA and 5 S genes is the interaction between the tetratricopeptide repeat (TPR)-containing subunit of TFIIIC (TFIIIC131) and the TFIIB-related subunit of TFIIIB (TFIIIB70/Brf1). A fragment of TFIIIC131 that contains the hydrophilic amino terminus and two TPR arrays, with five and four repeats, respectively (Nt-TPR9), is sufficient to support an interaction with TFIIIB70. Here we evaluate the contribution of each TPR array to TFIIIB70 binding. Both TPR arrays bind independently to TFIIIB70 with TPR6 -9 having a 4-fold higher apparent affinity than TPR1-5. However, the TPR arrays are not sufficient for a high affinity interaction with TFIIIB70. The addition of amino-terminal sequences increases the affinity of TPR1-5 18-fold to create a high affinity TFIIIB70 binding site (Nt-TPR5, 44 ؎ 6 nM). Although the Nt-TPR5 and TPR6 -9 fragments are contained entirely within the Nt-TPR9 fragment, the affinity of the latter is significantly lower than either of these smaller fragments. The results demonstrate that the TFIIIB70 binding sites in TFIIIC131 are subject to autoinhibition. We propose that the binding of TFIIIB70 to these sites within the TFIIIC complex may proceed in an ordered fashion.In eukaryotic cells, the transcription of 5 S rRNA, tRNA, and related genes by RNA polymerase III (pol III) 1 is dependent on transcription factor (TF) IIIC for promoter recognition and recruitment of the initiation factor TFIIIB to the DNA upstream of the transcription start site (1-3). In Saccharomyces cerevisiae, TFIIIC (also called ) is a large (ϳ520 kDa) six subunit complex (Ref. 4 and references therein) whose structure in electron micrographs reveals two flexibly linked globular domains (A and B) that interact, respectively, with the variably spaced internal A and B block promoter elements of tRNA genes (5, 6). The recruitment of TFIIIB by promoterbound TFIIIC is mediated initially by protein⅐protein interactions between the tetratricopeptide repeat (TPR)-containing subunit of TFIIIC (TFIIIC131) and the TFIIB-related subunit of TFIIIB (TFIIIB70/Brf1 (7-10)). Subsequently (or simultaneously) TBP is incorporated into the complex by protein⅐ protein interactions with TFIIIB70 and TFIIIC60 (4,9,11,12). These interactions serve to drive DNA binding by TBP (7). The assembly of TFIIIB is completed with the recruitment of TFIIIB90/BЉ by the other TFIIIB components and TFIIIC131

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