HEAT-SHOCK ELEMENT ARCHITECTURE IS AN IMPORTANT DETERMINANT IN THE TEMPERATURE AND TRANSACTIVATION DOMAIN REQUIREMENTS FOR HEAT-SHOCK TRANSCRIPTION FACTOR

The baker's yeast Saccharomyces cerevisiae possesses a single gene enc oding heat shock transcription factor (HSF), which is required for the activation of genes that participate in stress protection as well as normal growth and viability. Yeast HSF (yHSF) contains two distinct tr anscriptional activation regions located at the amino and carboxyl ter mini, Activation of the yeast metallothionein gene, CUP1, depends on a nonconsensus heat shock element (HSF), occurs at higher temperatures than other heat shock-responsive genes, and is highly dependent on the carboxyl-terminal transactivation domain (CTA) of yHSF, The results d escribed here show that the noncanonical (or gapped) spacing of GAA un its in the CUP1 HSE (HSE1) functions to limit the magnitude of CUP1 tr anscriptional activation in response to heat and oxidative stress. The spacing in HSE1 modulates the dependence for transcriptional activati on by both stresses on the yHSF CTA. Furthermore, a previously unchara cterized HSF in the CUP1 promoter, HSE2, modulates the magnitude of th e transcriptional activation of CUP1, via HSE1, in response to stress. In vitro DNase I footprinting experiments suggest that the occupation of HSE2 by yHSF strongly influences the manner in which yHSF occupies HSE1. Limited proteolysis assays show that HSF adopts a distinct prot ease-sensitive conformation when bound to the CUP1 HSE1, providing evi dence that the HSE influences DNA-bound HSF conformation. Together, th ese results suggest that CUP1 regulation is distinct from that of othe r classic heat shock genes through the interaction of yHSF with two no nconsensus HSEs, Consistent with this view, we have identified other g ene targets of yHSF containing HSEs with sequence and spacing features similar to those of CUP1 HSE1 and show a correlation between the spac ing of the GAA units and the relative dependence on the yHSF CTA.