TFIIIC plays a key role in nucleating the assembly of the initiation factor TFIIIB on class III genes. We have characterized an essential gene, TFC8, encoding the 60-kDa polypeptide, 60, present in affinity-purified TFIIIC. Hemagglutinin-tagged variants of 60 were found to be part of TFIIIC-tDNA complexes and to reside at least in part in the downstream DNA-binding domain B. Unexpectedly, the thermosensitive phenotype of N-terminally tagged 60 was suppressed by overexpression of 95, which belongs to the A domain, and by two TFIIIB components, TATA-binding protein (TBP) and B؆/TFIIIB90 (but not by TFIIIB70). Mutant TFIIIC was deficient in the activation of certain tRNA genes in vitro, and the transcription defect was selectively alleviated by increasing TBP concentration. Coimmunoprecipitation experiments support a direct interaction between TBP and 60. It is suggested that 60 links A and B domains and participates in TFIIIB assembly via its interaction with TBP.The primary step in tRNA gene activation is the binding of TFIIIC to the A and B blocks of the intragenic promoter. Its main function is then to assemble the initiation factor TFIIIB upstream of the transcription start site (30). Yeast TFIIIC ( factor) is a multisubunit protein of ca. 600 kDa organized in two large globular domains A and B of similar size and mass (10-nm diameter and ca. 300 kDa), each interacting with one promoter element, as visualized by electron microscopy (17, 53). Binding of B to the B block is predominant and favors the binding of A to the A block (4). TFIIIC-DNA interaction displays a remarkable adaptability to the variable A-B distances found in different tRNA genes (3). Affinity-purified Saccharomyces cerevisiae TFIIIC comprises six polypeptides of 138, 131, 95, 91,60, and 55 kDa (6,19,47,59). Gene cloning, protein-DNA cross-linking, mutagenesis, and protein-protein interaction studies provided a global view of the location and role of several TFIIIC subunits within the TFIIIC-DNA complex. 138 and 91 subunits reside in the B domain and cooperate in downstream DNA binding (1,10,36,37); 95 and 55 interact physically, belong to the A domain, and are thought to participate in A block binding (7,17,40,48,59). 131 (42) stands as the TFIIIC subunit responsible for TFIIIB assembly based on genetic evidence (49, 61), its upstream location (7), and its interaction with two TFIIIB components (14,33,51).TFIIIB is not a stable molecular entity like TFIIIC. It can be resolved chromatographically into two fractions named BЈ and BЉ (29). BЈ comprises TATA-binding protein (TBP) and the TFIIB-related factor TFIIIB70/Brf1 (12,16,31,39), while BЉ contains TFIIIB90 (32, 50, 51). The TFIIIC-dependent TFIIIB assembly on TATA-less class III genes is a multistep pathway that could be decomposed in vitro (29, 31) and reconstituted with recombinant TFIIIB components (32, 51). The order of interaction is TFIIIB70, then TBP, and then BЉ, as shown by gel retardation and DNA photo-cross-linking (31). TBP stabilizes the weak interaction between TFIIIB70 and th...