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 encoding 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 transcriptional activation regions located at the amino and carboxyl termini. Activation of the yeast metallothionein gene, CUP1, depends on a nonconsensus heat shock element (HSE), 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 described here show that the noncanonical (or gapped) spacing of GAA units in the CUP1 HSE (HSE1) functions to limit the magnitude of CUP1 transcriptional activation in response to heat and oxidative stress. The spacing in HSE1 modulates the dependence for transcriptional activation by both stresses on the yHSF CTA. Furthermore, a previously uncharacterized HSE in the CUP1 promoter, HSE2, modulates the magnitude of the 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 protease-sensitive conformation when bound to the CUP1 HSE1, providing evidence that the HSE influences DNA-bound HSF conformation. Together, these results suggest that CUP1 regulation is distinct from that of other classic heat shock genes through the interaction of yHSF with two nonconsensus HSEs. Consistent with this view, we have identified other gene targets of yHSF containing HSEs with sequence and spacing features similar to those of CUP1 HSE1 and show a correlation between the spacing of the GAA units and the relative dependence on the yHSF CTA.
Santoro
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Department of Biological Chemistry, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA.
Johansson
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Thiele
D J
DJ
D001483:Base Sequence; D002352:Carrier Proteins; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D005800:Genes, Fungal / Q000235:genetics; D006360:Heat-Shock Proteins; D008668:Metallothionein / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D018384:Oxidative Stress / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics
C500769:CUP1-1 protein, S cerevisiae; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C067168:HSF1 protein, S cerevisiae; D006360:Heat-Shock Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C007121:copper thionein; D008668:Metallothionein