Saturation mutagenesis of a yeast his3 "TATA element": genetic evidence for a specific TATA-binding protein.

Abstract

The yeast his3 promoter region contains two distinct classes of "TATA elements," constitutive (TC) and regulatory (TR), that are defined by their interactions with upstream promoter elements, selectivity of initiation sites, and chromatin structure. TC is localized between positions -83 and -53, and TR is localized between positions -55 and -35, regions in which there are several TATA-like sequences. In this study, we used saturation mutagenesis to examine the structural requirements of the his3 TR element necessary for transcriptional induction. To avoid the complications of redundant elements, the phenotypic analysis was carried out by using a gal-his3 hybrid promoter whose function depends on a short oligonucleotide containing the prospective his3 TR element. In this context, an oligonucleotide containing the sequence TATAAA is sufficient for TR function. However, 17 out of the 18 possible single-base substitutions and 9 out of 10 double mutations of this sequence abolish TR function. This strict sequence requirement for TR function strongly suggests that the TR element is a target site for a sequence-specific DNA-binding protein. Further, as the region encoding TC and promoters of certain other yeast genes do not contain a sequence that is compatible with TR function, we suggest that yeast cells contain multiple proteins with distinct sequence specificities that carry out a related "TATA function" and that yeast promoters can be divided into classes based on their downstream promoter elements.