Geraghty D S, Sucic H B, Chen J, Pederson D S
Department of Microbiology and Molecular Genetics and the Markey Center for Molecular Genetics, University of Vermont, Burlington, Vermont 05405-0068, USA.
J Biol Chem. 1998 Aug 7;273(32):20463-72. doi: 10.1074/jbc.273.32.20463.
In the yeast Saccharomyces cerevisiae, heat shock transcription factor (HSF) binds heat shock element (HSE) DNA shortly after DNA replication, independently of its activation by heat shock. To determine if HSF binding occurs before newly replicated DNA is packaged into nucleosomes, we inserted an HSE into a DNA segment that normally forms a positioned nucleosome in vivo. Transcription from constructs designed to create steric competition between binding of HSF and histone H2A-H2B dimers was generally poor, suggesting that nucleosome assembly precedes and inhibits HSF binding. However, one such construct was as transcriptionally active as a nucleosome-free control. Structural analyses suggested that approximately 40 base pairs of DNA, including the HSE, had unwrapped from the 3' edge of the histone octamer, allowing HSF to bind; approximately 100 base pairs remained in association with the histone octamer, with the same translational and rotational orientation as was seen for the poorly transcribed constructs. Modeling studies suggest that the active and inactive constructs differ from one another in the ease with which the HSE and flanking sequences can adopt the curvature needed to form a stable nucleosome. These differences may influence the probability of DNA unwrapping from already assembled nucleosomes and the subsequent binding of HSF.
在酿酒酵母中，热休克转录因子（HSF）在DNA复制后不久就会结合热休克元件（HSE）DNA，这一过程与热休克对其的激活无关。为了确定HSF的结合是否发生在新复制的DNA被包装成核小体之前，我们将一个HSE插入到一个在体内通常形成定位核小体的DNA片段中。旨在在HSF与组蛋白H2A-H2B二聚体结合之间产生空间竞争的构建体的转录通常很差，这表明核小体组装先于并抑制HSF结合。然而，其中一个这样的构建体的转录活性与无核小体对照一样高。结构分析表明，包括HSE在内的大约40个碱基对的DNA已从组蛋白八聚体的3'边缘解开，从而使HSF能够结合；大约100个碱基对仍与组蛋白八聚体结合，其平移和旋转方向与转录较差的构建体相同。建模研究表明，活性和非活性构建体在HSE及其侧翼序列形成稳定核小体所需曲率的难易程度上彼此不同。这些差异可能会影响DNA从已组装核小体上解开的概率以及随后HSF的结合。
