Sogawa K, Kikuchi Y, Imataka H, Fujii-Kuriyama Y
Department of Chemistry, Faculty of Science, Tohoku University, Sendai.
J Biochem. 1993 Oct;114(4):605-9. doi: 10.1093/oxfordjournals.jbchem.a124224.
We have expressed truncated forms of BTEB and Sp1 in Escherichia coli and investigated the DNA-binding properties of the two proteins. The two proteins as well as their chimeric proteins protected the same DNA region in the BTE sequence (a GC box in the P-4501A1 gene) as examined by ortho-phenanthroline-Cu footprinting. The region overlapped nearly perfectly with the GC box consensus sequence. Methylation interference footprinting revealed that all the guanines within the region and two other guanines in the close vicinity interacted with the proteins. Competitive gel mobility shift assay using various synthetic oligonucleotides of the GC box sequences as the competitors demonstrated that BTEB and Sp1 have similar sequence specificities for DNA binding. We have purified the bacterially expressed BTEB and measured the dissociation constant of the BTEB-BTE complex using gel mobility shift assay. The dissociation constant was (3.0 +/- 1.0) x 10(-10) M and was comparable to that of Sp1 binding to a GC box. Taken together, these findings indicate that the binding modes of BTEB and Sp1 to the GC box are similar to each other.
我们在大肠杆菌中表达了截短形式的BTEB和Sp1，并研究了这两种蛋白质的DNA结合特性。通过邻菲罗啉 - 铜足迹法检测发现，这两种蛋白质及其嵌合蛋白在BTE序列（P - 4501A1基因中的一个GC框）中保护相同的DNA区域。该区域与GC框共有序列几乎完全重叠。甲基化干扰足迹法表明，该区域内的所有鸟嘌呤以及附近的另外两个鸟嘌呤与蛋白质相互作用。使用各种GC框序列的合成寡核苷酸作为竞争者进行的竞争性凝胶迁移率变动分析表明，BTEB和Sp1对DNA结合具有相似的序列特异性。我们纯化了细菌表达的BTEB，并使用凝胶迁移率变动分析测量了BTEB - BTE复合物的解离常数。解离常数为(3.0 +/- 1.0) x 10(-10) M，与Sp1与GC框结合的解离常数相当。综上所述，这些发现表明BTEB和Sp1与GC框的结合模式彼此相似。
