Sigvardsson M, Leanderson T
Immunology Unit, Lund University, Sweden.
Mol Immunol. 1994 Sep;31(13):1005-16. doi: 10.1016/0161-5890(94)90095-7.
The SP6 kappa promoter decamer 3' flanking sequence was shown to contain several positive control elements for transcriptional stimulation which together increased transcriptional stimulation one order of magnitude. One was mapped to an A nucleotide immediately 3' of the decamer consensus motif. The mechanism was to increase the binding affinity of Oct2 for the decamer core motif by a direct interaction between the Oct2 protein and the template, since in vitro transcribed and translated Oct2 showed an identical binding preference when compared to the corresponding gene products in nuclear extracts. By mutation analysis it was shown that the functional activity of the SP6 kappa promoter 3' flanking sequence with regard to transcriptional stimulation was dependent on the presence of such a high affinity Oct binding site. Deletion analysis showed that the POU-Homeo domain of Oct2 sufficed for the flanking sequence mediated affinity increase for decamer elements and that amino or carboxy terminal variations in Oct2 did not influence this interaction. The flanking sequence element also increased Oct1 binding to the same decamer motif. The A 3' of the decamer increased affinity of Oct2 binding to a consensus as well as a mutated decamer. These data illustrate how flanking sequence elements can compensate mutations in the decamer core motif present in a majority of kappa promoters.
SP6 κ启动子十聚体3'侧翼序列被证明含有几个用于转录刺激的正调控元件，这些元件共同使转录刺激增加了一个数量级。其中一个元件被定位到十聚体共有基序3'端紧邻的一个A核苷酸。其机制是通过Oct2蛋白与模板之间的直接相互作用来增加Oct2对十聚体核心基序的结合亲和力，因为与核提取物中的相应基因产物相比，体外转录和翻译的Oct2显示出相同的结合偏好。通过突变分析表明，SP6 κ启动子3'侧翼序列在转录刺激方面的功能活性取决于这种高亲和力Oct结合位点的存在。缺失分析表明，Oct2的POU-同源结构域足以使侧翼序列介导的对十聚体元件的亲和力增加，并且Oct2的氨基或羧基末端变异不影响这种相互作用。侧翼序列元件也增加了Oct1与相同十聚体基序的结合。十聚体3'端的A增加了Oct2与共有十聚体以及突变十聚体结合的亲和力。这些数据说明了侧翼序列元件如何能够补偿大多数κ启动子中存在的十聚体核心基序中的突变。
