Zhang J, Jacob S T
Department of Pharmacology and Molecular Biology, Chicago Medical School, Illinois 60064-3095.
Mol Cell Biol. 1990 Oct;10(10):5177-86. doi: 10.1128/mcb.10.10.5177-5186.1990.
Previous studies in our laboratory have characterized a 174-base-pair (bp) enhancer sequence in the rat ribosomal DNA spacer region that exhibits all of the characteristics of a polymerase (Pol) II enhancer. Further studies showed that at least half of the enhancer activity resides in a 37-bp motif (E1) within the 174-bp spacer sequence that is located between positions -2.183 and -2.219 kilobase pairs upstream of the initiation site. To identify the factor(s) that binds specifically to the 37-bp enhancer domain, we fractionated whole-cell extract from rat adenocarcinoma ascites cells by chromatography on a series of columns, including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides of molecular weights 79,400 and 89,100 and was completely devoid of RNA Pol I activity. Electrophoretic mobility shift analysis showed that the polypeptides in the purified preparation (designated E1BF) interacted with both the enhancer element and the core promoter. To determine whether each polypeptide can separately bind to the core promoter and the enhancer, the individual components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, renatured, and subjected to gel retardation analysis. This experiment demonstrated that both polypeptides interacted with the two cis-acting sequences. The specificity of the binding was demonstrated by competition with unlabeled 37-bp and core promoter fragments and lack of competition with nonspecific DNAs in the mobility shift assay. The 37-bp enhancer as well as the downstream sequence of the core promoter were protected by E1BF in the DNase I footprinting assay. Addition of E1BF to limiting amounts of fraction DE-B, which contains all factors essential for Pol I-directed transcription, resulted in three- to fourfold stimulation of ribosomal DNA transcription. Comparison of molecular weights and footprinting profiles did not reveal any relationship between E1BF and other Pol I trans-acting factors.
我们实验室之前的研究已经对大鼠核糖体DNA间隔区中一段174个碱基对（bp）的增强子序列进行了表征，该序列展现出了聚合酶（Pol）II增强子的所有特征。进一步的研究表明，至少一半的增强子活性存在于174 bp间隔序列内的一个37 bp基序（E1）中，该基序位于起始位点上游-2.183至-2.219千碱基对之间。为了鉴定与37 bp增强子结构域特异性结合的因子，我们通过在一系列柱上进行色谱分离，包括寡脱氧核苷酸亲和柱，对大鼠腺癌腹水细胞的全细胞提取物进行了分级分离。最终制剂包含分子量分别为79,400和89,100的两种多肽，并且完全没有RNA Pol I活性。电泳迁移率变动分析表明，纯化制剂中的多肽（命名为E1BF）与增强子元件和核心启动子都相互作用。为了确定每种多肽是否能分别与核心启动子和增强子结合，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳将各个组分分离，复性，然后进行凝胶阻滞分析。该实验表明两种多肽都与这两个顺式作用序列相互作用。结合特异性通过与未标记的37 bp和核心启动子片段竞争以及在迁移率变动试验中与非特异性DNA缺乏竞争来证明。在DNase I足迹试验中，37 bp增强子以及核心启动子的下游序列受到E1BF的保护。向含有Pol I指导转录所需所有因子的限量级分DE-B中添加E1BF，导致核糖体DNA转录受到三到四倍的刺激。分子量和足迹图谱的比较未揭示E1BF与其他Pol I反式作用因子之间的任何关系。
