A novel upstream RNA polymerase III promoter element becomes essential when the chromatin structure of the yeast U6 RNA gene is altered

The Saccharomyces cerevisiae U6 RNA gene, SNR6, possesses upstream sequence s that allow productive binding in vitro of the RNA polymerase III (Pol III ) transcription initiation factor IIIB (TFIIIB) in the absence of TFIIIC or other assembly factors. TFIIIC-independent transcription of SNR6 in vitro is highly sensitive to point mutations in a consensus TATA box at position -30. In contrast, the TATA box is dispensable for SNR6 transcription in viv o, apparently because TFIIIC bound to the intragenic A block and downstream B block can recruit TFIIIB via protein-protein interactions. A mutant alle le of SNR6 with decreased spacing between the A and B blocks, snr6-Delta 42 , exhibits increased dependence on the upstream sequences in vivo. Unexpect edly, we find that in vivo expression of snr6-Delta 42 is much more sensiti ve to mutations in a (dT-dA)(7) tract between the TATA box and transcriptio n start site than to mutations in the TATA box itself. Inversion of single base pairs in the center of the dT-dA tract nearly abolishes transcription of snr6-Delta 42, yet inversion of all 7 base pairs has little effect on ex pression, indicating that the dA-dT tract is relatively orientation indepen dent. Although it is within the TFIIIB footprint, point mutations in the dT -dA tract do not inhibit TFIIIB binding or TFIIIC-independent transcription of SNR6 in vitro. In the absence of the chromatin architectural protein Nh p6, dT-dA tract mutations are lethal even when A-to-B block spacing is wild type. We conclude that the (dT-dA)(7) tract and Nhp6 cooperate to direct p roductive transcription complex assembly on SNR6 in vivo.