ANALYSIS OF SACCHAROMYCES-CEREVISIAE HIS3 TRANSCRIPTION INVITRO - BIOCHEMICAL SUPPORT FOR MULTIPLE MECHANISMS OF TRANSCRIPTION

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, T(C) and T(R), that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that T(C) and T(R) promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of T(R) derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the T(R) element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of T(C) function in vitro. The results support the idea that T(C) and T(R) mediate transcription from the wild-type promoter by distinct mechanisms.