Saltsman K A, Prentice H L, Kingston R E
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114, USA.
Yeast. 1998 Jun 15;14(8):733-46. doi: 10.1002/(SICI)1097-0061(19980615)14:8<733::AID-YEA270>3.0.CO;2-8.
The C-terminal hydrophobic repeat (CTR) of heat shock transcription factor (HSF) has been proposed to regulate DNA binding by intramolecular interactions with the leucine zipper motifs present in the HSF trimerization domain. Schizosaccharomyces pombe provides a useful model organism for the study of the regulation of HSF DNA binding because, unlike Saccharomyces cerevisiae, S. pombe hsf is highly heat shock inducible for DNA binding and contains a clear homology to the CTR. We examined the role that the CTR plays in the regulation of S. pombe hsf by constructing isogenic strains bearing deletion and point mutations in the chromosomal copy of hsf. Surprisingly, we found that point mutation of key hydrophobic amino acids within the CTR, as well as full deletion of it, yielded factors that show normal binding at normal growth temperatures and full levels of heat-induced binding. Deletion of the CTR did, however, slightly lower the temperature required for maximal activation. In contrast, a large deletion of the C-terminus, which removes close to a third of the coding sequence, was deregulated and bound DNA at control temperature. Several of the deletion mutants were significantly reduced in their level of expression, yet they showed wild-type levels of DNA binding activity following heat shock. These experiments demonstrate that appropriate regulation of the DNA binding activity of S. pombe hsf is not solely dependent upon the CTR, and imply that a feedback mechanism exists that establishes proper levels of DNA binding following heat shock despite mutations that significantly alter levels of total hsf.
热休克转录因子（HSF）的C末端疏水重复序列（CTR）被认为可通过与HSF三聚化结构域中存在的亮氨酸拉链基序进行分子内相互作用来调节DNA结合。粟酒裂殖酵母为研究HSF DNA结合的调控提供了一种有用的模式生物，因为与酿酒酵母不同，粟酒裂殖酵母hsf对DNA结合具有高度热休克诱导性，并且与CTR具有明显的同源性。我们通过构建在hsf染色体拷贝中带有缺失和点突变的同基因菌株，研究了CTR在粟酒裂殖酵母hsf调控中所起的作用。令人惊讶的是，我们发现CTR内关键疏水氨基酸的点突变以及CTR的完全缺失产生的因子在正常生长温度下显示正常结合，并且热诱导结合水平完全正常。然而，CTR的缺失确实略微降低了最大激活所需的温度。相比之下，C末端的大量缺失（去除了近三分之一的编码序列）则不受调控，并在对照温度下结合DNA。几个缺失突变体的表达水平显著降低，但在热休克后它们显示出野生型水平的DNA结合活性。这些实验表明，粟酒裂殖酵母hsf的DNA结合活性的适当调控并不完全依赖于CTR，这意味着存在一种反馈机制，尽管突变显著改变了总hsf的水平，但在热休克后仍能建立适当水平的DNA结合。
