In yeast, heat shock factor (HSF) is a trimer that binds DNA constitutively but only supports high levels of transcription upon heat shock. The C-terminal regions of HSF from Saccharomyces cerevisiae and Kluyveromyces lactis are unconserved yet both contain strong transactivators which are correctly regulated when substituted for each other. We have performed high resolution mapping of these activator domains which shows that in K.lactis HSF (KlHSF) activity can be located to a confined short domain, while in S.cerevisiae HSF (ScHSF) two separate regions are required for full activity. Alignment of the activator domains reveals similarity, as both overlap potential leucine zipper motifs (zipper C) with a distribution of hydrophobic residues similar to two highly conserved N-terminal domains which mediate HSF trimerization (zippers A and B). In higher eukaryotes a C-terminal leucine zipper is required to maintain HSF in a monomeric and non DNA-binding state under normal conditions and we therefore address the regulatory roles of the three leucine zipper motifs in KlHSF. Whilst the longest and most N-terminal of the trimer region zippers, A, is dispensable for regulation, mutation of a single leucine in zipper B makes HSF constitutively active. In contrast to the situation in higher eukaryotes disruption of zipper C has no observable regulatory effect and therefore, although an intramolecular contact between zippers B and C cannot be ruled out, such contact is not required for restraining the C-terminal activator domain. We furthermore find that deletions which abolish activator potential of the C-terminus render the host strain temperature sensitive. However, deletion of a double proline-glycine motif in the activator, whilst leaving HSF unable to respond to heat shock, does not cause temperature sensitivity. This result demonstrates that independent mechanisms control the transient and sustained activities of HSF.

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Chen Y, Barlev NA, Westergaard O, Jakobsen BK

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