Identification of transcriptional activation and inhibitory domains in serum response factor (SRF) by using GAL4-SRF constructs.
Identification of transcriptional activation and inhibitory domains in serum response factor (SRF) by using GAL4-SRF constructs.
复制标题
使用 GAL4-SRF 构建体鉴定血清反应因子 (SRF) 中的转录激活和抑制结构域。
DOI：
10.1128/mcb.13.8.4640-4647.1993
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发表时间：
1993
影响因子：
5.3
通讯作者：
Prywes,R
中科院分区：
文献类型：
--
作者：
Johansen,FE;Prywes,R
血清反应因子(SRF)与c-fos血清反应元件的结合是血清和生长因子激活c-fos所必需的。由于SRF在细胞中广泛表达，因此很难对其活性进行测量。为了在细胞中检测SRF的功能，我们通过将其与GAL4的DNA结合域融合来改变其DNA结合特异性。将GAL4-SRF结构物导入细胞，使我们能够确定SRF的转录激活结构域以及抑制该活性的结构域。首先，我们发现转录激活区域在HeLa细胞中位于339-508位氨基酸之间，在NIH 3T3细胞中位于414-508位氨基酸之间。其次，我们证明在GAL4-SRF构建的背景下，SRF有两个单独的结构域可以抑制其激活结构域。尽管这些结构域与SRF的DNA结合和二聚化结构域重叠，但这些功能并不是抑制所必需的。最后，我们证明了其中一个抑制结构域是模块化的，当它的氨基末端移动到SRF-Gal4-SRF中的Gal4‘S DNA结合域时，它也可以抑制激活。讨论了这些抑制结构域对SRF调节的影响。
The binding of serum response factor (SRF) to the c-fos serum response element has been shown to be essential for serum and growth factor activation of c-Fos. Since SRF is ubiquitously expressed, it has been difficult to measure the activity of SRF introduced into cells. To assay for functions of SRF in cells, we have changed its DNA binding specificity by fusing it to the DNA binding domain of GAL4. Transfection of GAL4-SRF constructs into cells has allowed us to identify SRF's transcriptional activation domain as well as domains which inhibit this activity. First, we found that the transcriptional activation domain maps to between amino acids 339 and 508 in HeLa cells and to between amino acids 414 and 508 in NIH 3T3 cells. Second, we show that in the context of GAL4-SRF constructs, there are two separate domains of SRF that can inhibit its activation domain. Although these domains overlap the DNA binding and dimerization domains of SRF, these functions were not required for inhibition. Finally, we show that one of the inhibitory domains is modular in that it can also inhibit activation when it is moved amino terminal to GAL4's DNA binding domain in an SRF-GAL4-SRF construct. The implications of these inhibitory domains for SRF regulation are discussed.