Nieto-Sotelo J, Ichida A, Quail P H
University of California-Berkeley/United States Department of Agriculture, Plant Gene Expression Center, Albany 94710.
Plant Cell. 1994 Feb;6(2):287-301. doi: 10.1105/tpc.6.2.287.
Phytochrome-imposed down-regulation of the expression of its own phytochrome A gene (PHYA) is one of the fastest light-induced effects on transcription reported in plants to date. Functional analysis of the oat PHYA3 promoter in a transfection assay has revealed two positive elements, PE1 and PE3, that function synergistically to support high levels of transcription in the absence of light. We have isolated a rice cDNA clone (pR4) encoding a DNA binding protein that binds to the AT-rich PE1 element. We tested the selectivity of the pR4-encoded DNA binding activity using linker substitution mutations of PE1 that are known to disrupt positive expression supported by the PHYA3 promoter in vivo. Binding to these linker substitution mutants was one to two orders of magnitude less than to the native PE1 element. Because this is the behavior expected of positive factor 1 (PF1), the presumptive nuclear transcription factor that acts in trans at the PE1 element in vivo, the data support the conclusion that the protein encoded by pR4 is in fact rice PF1. The PF1 polypeptide encoded by pR4 is 213 amino acids long and contains four repeats of the A-T hook DNA binding motif found in high-mobility group I-Y (HMGI-Y) proteins. In addition, PF1 contains an 11-amino acid-long hydrophobic region characteristic of HMG I proteins, its N-terminal region shows strong similarities to a pea H1 histone sequence and a short peptide sequence from wheat HMGa, and it shows a high degree of similarity along its entire length to the HMG Y-like protein encoded by a soybean cDNA, SB16. In vitro footprinting and quantitative gel shift analyses showed that PF1 binds preferentially to the PE1 element but also at lower affinity to two other AT-rich regions upstream of PE1. This feature is consistent with the binding characteristics of HMG I-Y proteins that are known to bind to most runs of six or more AT base pairs. Taken together, the properties of PF1 suggest that it belongs to a newly described family of nuclear proteins containing both histone H1 domains and A-T hook DNA binding domains.
光敏色素对其自身光敏色素A基因（PHYA）表达的负调控是迄今为止植物中报道的最快的光诱导转录效应之一。在转染实验中对燕麦PHYA3启动子进行功能分析，发现了两个正向元件PE1和PE3，它们在无光条件下协同作用以支持高水平转录。我们分离出一个水稻cDNA克隆（pR4），其编码一种与富含AT的PE1元件结合的DNA结合蛋白。我们使用已知会破坏体内PHYA3启动子支持的正向表达的PE1连接子取代突变体，测试了pR4编码的DNA结合活性的选择性。与这些连接子取代突变体的结合比与天然PE1元件的结合少一到两个数量级。由于这是体内在PE1元件上反式作用的推定核转录因子正向因子1（PF1）预期的行为，这些数据支持了pR4编码的蛋白实际上是水稻PF1的结论。pR4编码的PF1多肽长213个氨基酸，包含在高迁移率族I-Y（HMGI-Y）蛋白中发现的A-T钩DNA结合基序的四个重复序列。此外，PF1包含HMG I蛋白特有的11个氨基酸长的疏水区域，其N端区域与豌豆H1组蛋白序列和小麦HMGa的一个短肽序列有很强的相似性，并且它在全长上与大豆cDNA编码的HMG Y样蛋白SB16有高度相似性。体外足迹分析和定量凝胶迁移分析表明，PF1优先与PE1元件结合，但也以较低亲和力与PE1上游的另外两个富含AT的区域结合。这一特征与已知能结合大多数六个或更多AT碱基对连续序列的HMG I-Y蛋白的结合特性一致。综合来看，PF1的特性表明它属于一个新描述的核蛋白家族，该家族同时包含组蛋白H1结构域和A-T钩DNA结合结构域。
