TFIIIB PLACEMENT ON A YEAST U6 RNA GENE IN-VIVO IS DIRECTED PRIMARILYBY TFIIIC RATHER THAN BY SEQUENCE-SPECIFIC DNA CONTACTS

The Saccharomyces cerevisiae U6 RNA gene (SNR6), which is transcribed by RNA polymerase LII, has an unusual combination of promoter elements : an upstream TATA box, an intragenic A block, and a downstream B bloc k In tRNA genes, the A and B blocks are binding sites for the transcri ption initiation factor TFIIIC, which positions TFIIIB a fixed distanc e upstream of the A block However, in vitro transcription of SNR6 with purified components requires neither TFIIIC nor the A and B blocks, p resumably because TPIIIB recognizes the upstream sequences directly. H ere we demonstrate that TFIIIB placement on SNR6 in vivo is directed p rimarily by the TFIIIC-binding elements rather than by upstream sequen ces. We show that the A block is a stronger start site determinant tha n the upstream sequences when the two are uncoupled by an insertion mu tation. Furthermore, while TFIIIC-independent in vitro transcription o f SNR6 is highly sensitive to TATA box point mutations, in vivo initia tion on SNR6 is only marginally sensitive to such mutations unless the A block is mutated. Intriguingly, a deletion downstream of the U6 RNA coding region that reduces A-to-B block spacing also increases in viv o dependence on the TATA box. Moreover, this deletion results in the a ppearance of micrococcal nuclease-hypersensitive sites in the TFIIIB c hromatin footprint, indicating that TFIIIB binding is disrupted by a m utation 150 bp distant. This and additional chromatin footprinting dat a suggest that SNR6 is assembled into a nucleoprotein complex that fac ilitates the TFIIIC-dependent binding of TFIIIB.