Lee M, Song H, Park S, Park J
Department of Chemistry, Seoul National University, Korea.
Biol Chem. 1998 Nov;379(11):1333-40. doi: 10.1515/bchm.1998.379.11.1333.
In this study we analyzed the ratp53 promoter by electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis. As a result we identified two protein binding elements (element 1: -296 to -312, element 2: -195 to -219) with sequence homology to each other. The two identified elements bind to the same kind of protein. To identify the protein binding to these elements, competition assays were carried out with double stranded oligonucleotides containing NF1, YY1, and CRE consensus motifs. Only the NF1 consensus motif competed with element 1 and 2. Element 2 is conserved between the rat, human, and mouse p53 promoters, and has an NF1 consensus motif. However, the sequences of element 1 are comparatively variable between the species. Only the element 1 region of the rat p53 promoter has partial homology to the NF1 consensus motif. This suggests that the element 1 is specific for the rat p53 gene. The molecular mass of the binding protein, determined by Southwestern blotting analysis, was 40 kDa, which is different from that of NF1. In EMSA with an anti-NF1 antibody, DNA-protein complexes were neither supershifted nor decreased. The 40 kDa protein was also detected in rat spleen and lung, but not in kidney. The binding protein was purified by sequence-specific DNA affinity chromatography and it was confirmed that the purified protein binds to the two regions. It was also proved that the identified two elements are required for basal level transcription of the rat p53 gene by in vitro transcription assay.
在本研究中，我们通过电泳迁移率变动分析（EMSA）和DNase I足迹分析对大鼠p53启动子进行了分析。结果，我们鉴定出两个彼此具有序列同源性的蛋白质结合元件（元件1：-296至-312，元件2：-195至-219）。所鉴定的这两个元件与同一种蛋白质结合。为了鉴定与这些元件结合的蛋白质，我们用含有NF1、YY1和CRE共有基序的双链寡核苷酸进行了竞争试验。只有NF1共有基序能与元件1和元件2竞争。元件2在大鼠、人类和小鼠p53启动子之间保守，并且具有一个NF1共有基序。然而，元件1的序列在不同物种之间相对可变。大鼠p53启动子的元件1区域仅与NF1共有基序有部分同源性。这表明元件1对大鼠p53基因具有特异性。通过蛋白质免疫印迹分析确定的结合蛋白的分子量为40 kDa，这与NF1的分子量不同。在使用抗NF1抗体的EMSA中，DNA-蛋白质复合物既没有发生超迁移也没有减少。在大鼠脾脏和肺中也检测到了40 kDa的蛋白质，但在肾脏中未检测到。通过序列特异性DNA亲和色谱法纯化了结合蛋白，并证实纯化后的蛋白与这两个区域结合。体外转录试验也证明，所鉴定的这两个元件是大鼠p53基因基础水平转录所必需的。
