H Rudolph's scientific contributions

Transcription of PHO5 is strongly regulated in response to the level of inorganic phosphate (Pi) present in the growth medium. We have identified elements required for PHO5 expression by analyzing small deletions in the PHO5 promoter on chromosome II. The results reveal three functionally different components of the PHO5 promoter: regulatory regions, a "TATA" element, and specific mRNA initiation sites. The regulatory regions contain related 19-base-pair (bp) dyad sequences acting as phosphate-controlled upstream activation sites (UASpS). These UASpS mediate the transcriptional activation of PHO5 observed in low Pi conditions. The unlinked but coordinately regulated PHO11 promoter contains a single copy of an almost identical dyad sequence, suggesting that there is a common regulatory UASp for both genes. A TATA element is absolutely required for detectable PHO5 transcription. Specific purine-pyrimidine motifs (RRYRR) (R = purine and Y = pyrimidine) serve as PHO5 mRNA initiation sites, but only if they lie 55-110 bp downstream of a functional TATA element. Such an "initiation window" is not found in higher eukaryotes and implies mechanistic differences in the transcription machineries between yeast and higher eukaryotes.

The chromatin fine structure in the promoter region of PHO5, the structural gene for a strongly regulated acid phosphatase in yeast, was analyzed. An upstream activating sequence 367 bp away from the start of the coding sequence that is essential for gene induction was found to reside in the center of a hypersensitive region under conditions of PHO5 repression. Under these conditions three related elements at positions -469, -245 and -185 are contained within precisely positioned nucleosomes located on both sides of the hypersensitive region. Upon PHO5 induction the chromatin structure of the promoter undergoes a defined transition, in the course of which two nucleosomes upstream and two nucleosomes downstream of the hypersensitive site are selectively removed. In this way approximately 600 bp upstream of the PHO5 coding sequence become highly accessible and all four elements are free to interact with putative regulatory proteins. These findings suggest a mechanism by which the chromatin structure participates in the functioning of a regulated promoter.