Carlson T, Christian N, Bonner J J
Department of Biology, Indiana University, Bloomington 47405, USA.
Gene Expr. 1999;7(4-6):283-91.
Yeast HSF is constitutively trimeric and DNA bound. Heat shock is thought to activate HSF by inducing a conformational change. We have developed an assay in which we can follow a conformational change of HSF that correlates with activity and thus appears to be the active conformation. This conformational change requires two HSF trimers bound cooperatively to DNA. The conformational change can be induced in whole cell extracts, and is thus amenable to biochemical analysis. We have purified a factor that triggers the conformational change. The factor is sensitive to dialysis, insensitive to NEM, and is not extractable by phenol. It is small, and apparently not a peptide. Mass spectroscopy identifies a novel guanine nucleotide that tracks with activity on columns. This novel nucleotide, purchased from Sigma, induces the conformational change (although this does not prove the identity of the activating factor unambiguously, because Sigma's preparation is contaminated with other compounds). What is the source of this nucleotide in cells? Activity can be generated by treating extracts with ribonuclease; this implicates RNA degradation as a source of HSF-activating activity. The heat shock response is primarily responsible for monitoring the levels of protein chaperones; how can RNA degradation be involved? Synthetic lethal interactions link HSF activity to ribosome biogenesis, suggesting a possible model. Ribosomal proteins are produced in large quantities, and in excess of rRNA; unassembled r-proteins are rapidly degraded (t1/2 approximately 3 min). Unassembled r-proteins aggregate readily. It is likely that unassembled r-proteins represent a major target of chaperones in vivo, and for proteasome-dependent degradation. Interference with rRNA processing (e.g., by heat shock) requires hsp70s to handle the aggregation-prone r-proteins, and proteasome proteins to help degrade the unassembled r-proteins before they aggregate. A nucleotide signal could be generated from the degradation products of the rRNA itself.
酵母热休克因子（HSF）以三聚体形式组成性地与DNA结合。热休克被认为通过诱导构象变化来激活HSF。我们开发了一种检测方法，通过该方法可以追踪与活性相关且似乎是活性构象的HSF构象变化。这种构象变化需要两个HSF三聚体协同结合到DNA上。这种构象变化可以在全细胞提取物中诱导产生，因此适合进行生化分析。我们已经纯化了一种触发构象变化的因子。该因子对透析敏感，对N - 乙基马来酰亚胺（NEM）不敏感，且不能用苯酚提取。它体积小，显然不是肽。质谱分析鉴定出一种与柱上活性相关的新型鸟嘌呤核苷酸。这种从西格玛公司购买的新型核苷酸可诱导构象变化（尽管这并不能明确证明激活因子的身份，因为西格玛公司的制剂被其他化合物污染）。细胞中这种核苷酸的来源是什么？用核糖核酸酶处理提取物可以产生活性；这表明RNA降解是HSF激活活性的一个来源。热休克反应主要负责监测蛋白质伴侣的水平；RNA降解是如何参与其中的呢？合成致死相互作用将HSF活性与核糖体生物发生联系起来，提示了一种可能的模型。核糖体蛋白大量产生，且超过rRNA的量；未组装的核糖体蛋白会迅速降解（半衰期约为3分钟）。未组装的核糖体蛋白很容易聚集。未组装的核糖体蛋白很可能是体内伴侣蛋白以及蛋白酶体依赖性降解的主要靶点。干扰rRNA加工（例如通过热休克）需要热休克蛋白70（hsp70s）来处理易于聚集的核糖体蛋白，以及蛋白酶体蛋白来帮助在未组装的核糖体蛋白聚集之前将其降解。一种核苷酸信号可能由rRNA本身的降解产物产生。
