THE SYMMETRY OF THE YEAST U6 RNA GENES TATA BOX AND THE ORIENTATION OF THE TATA-BINDING PROTEIN IN YEAST TFIIIB

The central RNA polymerase III (Pol III) transcription factor TFIIIB i s composed of the TATA-binding protein (TBP), Brf, a protein related t o TFIIB, and the product of the newly cloned TFC5 gene. TFIIIB assembl es autonomously on the upstream promoter of the yeast U6 snRNA (SNR6) gene in vitro, through the interaction of its TBP subunit with a conse nsus TATA box located at base pair -30. As both the DNA-binding domain of TBP and the U6 TATA box are nearly twofold symmetrical, we have ex amined how the binding polarity of TEIIIB is determined. We find that TFIIIB can bind to the U6 promoter in both directions, that TBP is una ble to discern the natural polarity of the TATA element and that, as a consequence, the U6 TATA box is functionally symmetrical. A modest pr eference for TFIIIB binding in the natural direction of the U6 promote r is instead dictated by flanking DNA. Because the assembly of TFIIIB on the yeast U6 gene in vivo occurs via a TEIIIC-dependent mechanism, we investigated the influence of TFIIIC on the binding polarity of TFI IIB. TFIIIC places TFIIIB on the promoter in one direction only; thus, it is TFIIIC that primarily specifies the direction of transcription. Experiments using TFIIIB reconstituted with the altered DNA specifici ty mutant TBPm3 demonstrate that in the TFIIIB-U6 promoter complex, th e carboxy-terminal repeat of TBP contacts the upstream half of the TAT A box. This orientation of yeast TBP in pol III promoter-bound TFIIIB is the same as in pol II promoter-bound TFIID and in TBP-DNA complexes that have been analyzed by X-ray crystallography.