Ordering promoter binding of class III transcription factors TFIIIC1 and TFIIIC2

The separation of the mammalian class III transcription factor TFIIIC into two functional components, termed TFIIIC1 and TFIIIC2, enabled an analysis of their functions in transcription initiation. Template competition assays were used to define the order with which these factors interact in vitro to form stable preinitiation complexes on the adenovirus VAI and Drosophila melanogaster tRNA(Arg) genes. The interaction between these genes and TFIIIC2, the factor that binds with high affinity to the B block, was both necessary and sufficient for template commitment. When either the VAI or tRNA(Arg) gene was preincubated with TFIIIC2 alone, transcription of a second gene added subsequently was excluded, indicating that TFIIIC2 bound stably to the first template. Furthermore, the interaction between TFIIIC2 and these genes must occur prior to that of TFIIIC1 or TFIIIB. Once TFIIIC2 was bound, TFIIIC1 could bind to the tRNA(Arg) and VAI genes, although its interaction with the VAI gene was less stable than that with the tRNA(Arg) gene. TFIIIB activity bound stably to the complex of both genes with TFIIIC2. These results demonstrate that TFIIIC2 is the first transcription factor to bind to these genes and that TFIIIB and TFIIIC1 can then interact in either order to form a preinitiation complex.