Kondrashov A V, Pospelov V A
Institute of Cytology RAS, 194064 St. Petersburg, Russia.
Tsitologiia. 2001;43(8):764-71.
The aim of this work is to study in vitro mechanisms of interactions between the promoter and enhancer complexes of the transcriptional apparatus. We used retardation assay for the labeled TATA-box containing oligonucleotide with an unfractionated nuclear extract and with a second unlabeled oligonucleotide carrying an enhancer sequence. As an enhancer sequence we have chosen e5 element that is known to interact with members of Pax family transcription factors. We showed that the presence of unlabeled e5 element led to a significant decrease in the mobility of TATA-box associated complex. E5 element with a mutated homeodomen binding site does not produce such an effect, while a mutation in the paired binding site does not change the ability of e5 element to influence the formation of promoter-associated complex. We observed the same complex using crude nuclear extracts from cells that express chimeric protein Pax7-FKHR with higher molecular weight. The fact that the increase in the size of the transcription factor does not influence the size of the TATA-box associated complex led us to conclude that this complex did not result from association of original promoter and enhancer components, but rather the role of enhancer may be to stabilize the slowly migrating complex on the TATA-box via short-term interactions inducing conformational changes in the included proteins.
这项工作的目的是研究转录装置的启动子和增强子复合物之间相互作用的体外机制。我们使用含有标记TATA框的寡核苷酸与未分级核提取物以及携带增强子序列的第二种未标记寡核苷酸进行阻滞分析。作为增强子序列，我们选择了已知与Pax家族转录因子成员相互作用的e5元件。我们发现未标记的e5元件的存在导致TATA框相关复合物的迁移率显著降低。具有突变同源结构域结合位点的e5元件不会产生这种效应，而配对结合位点的突变不会改变e5元件影响启动子相关复合物形成的能力。我们使用表达更高分子量嵌合蛋白Pax7-FKHR的细胞的粗核提取物观察到了相同的复合物。转录因子大小的增加不影响TATA框相关复合物的大小这一事实使我们得出结论，该复合物不是由原始启动子和增强子成分的结合产生的，相反，增强子的作用可能是通过短期相互作用诱导所含蛋白质的构象变化，从而稳定TATA框上缓慢迁移的复合物。
