A 100-kD HeLa cell octamer binding protein (OBP100) interacts differently with two separate octamer-related sequences within the SV40 enhancer.
A 100-kD HeLa cell octamer binding protein (OBP100) interacts differently with two separate octamer-related sequences within the SV40 enhancer.
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DOI：
10.1101/gad.1.10.1147
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发表时间：
1987-12
影响因子：
10.5
通讯作者：
Richard Sturm;T. Baumruker;B. R. Franza;W. Herr
中科院分区：
文献类型：
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作者：
Richard Sturm;T. Baumruker;B. R. Franza;W. Herr
许多真核生物上游启动子和增强子区域包含一个功能八聚体序列ATGCAAAT。我们研究了从HeLa细胞分离的八聚体结合蛋白与SV40和免疫球蛋白重链（IgH）基因增强子之间的相互作用。在凝胶阻滞实验中，部分纯化的八聚体结合活性与IgH增强子八聚体形成单一复合物，但与含有单个72-bp元件的SV40增强子片段形成两个复合物。通过使用点突变体和硫酸二甲酯和焦碳酸二乙酯修饰干扰实验，我们发现SV40复合物是由一个因子与八聚体相关序列ATGCAAAG （Octa1）和相邻的先前未识别的八聚体相关序列ATGCATCT （Octa2）结合而产生的。与Octa1和Octa2的碱基特异性相互作用不同；在10bp序列TATGCAAAGC上的化学修饰影响Octa1的结合，而在13bp序列ATGCATCTCAATT上的化学修饰影响Octa2的结合，其中八聚体样序列不在中心。通过DNA亲和沉淀法和sds -聚丙烯酰胺凝胶电泳对八聚体的结合活性进行了广泛的纯化。纯化后的蛋白OBP100表观分子量为100 kD，可结合SV40 Octa1和Octa2以及IgH增强子。OBP100与不同大小的Octa1和Octa2结合位点的不同相互作用表明OBP100与其结合位点之间具有非常灵活的序列识别。
Numerous eukaryotic upstream promoter and enhancer regions contain a functional octamer sequence ATGCAAAT. We have examined the interactions between an octamer binding protein isolated from HeLa cells and the SV40 and immunoglobulin heavy-chain (IgH) gene enhancers. A partially purified octamer binding activity forms a single complex with the IgH enhancer octamer in a gel retardation assay, but two complexes with a SV40 enhancer fragment containing a single 72-bp element. By using point mutants and both dimethyl sulfate and diethyl pyrocarbonate modification interference assays, we show that the SV40 complexes result from binding of a factor to the octamer-related sequence ATGCAAAG (Octa1) and to an adjacent previously unidentified octamer-related sequence ATGCATCT (Octa2). The base-specific interactions with Octa1 and Octa2 differ; chemical modifications over a 10-bp sequence TATGCAAAGC affect Octa1 binding whereas Octa2 binding is affected by modifications spanning a 13-bp sequence ATGCATCTCAATT in which the octamer-like sequence is not centered. The octamer binding activity has been purified extensively by a DNA affinity precipitation procedure and SDS-polyacrylamide gel electrophoresis. The purified protein, OBP100, has an apparent molecular weight of 100 kD and binds both SV40 Octa1 and Octa2, as well as the IgH enhancer. The distinct interactions of OBP100 with the differently sized Octa1 and Octa2 binding sites suggest remarkably flexible sequence recognition between OBP100 and its binding sites.