Yang Edward, Henriksen Melissa A, Schaefer Olaf, Zakharova Natalia, Darnell James E
Laboratory of Molecular Cell Biology, The Rockefeller University, New York, New York 10021-6399, USA.
J Biol Chem. 2002 Apr 19;277(16):13455-62. doi: 10.1074/jbc.M112038200. Epub 2002 Feb 7.
The STAT1 transcription factor is organized into several highly conserved domains, each of which has been assigned a function with the exception of the linker domain. We previously characterized a mutant in the linker domain of STAT1 that gave normal DNA binding using a standard probe in an electrophoretic mobility assay but failed to activate transcription in response to interferon gamma. We now report the mechanistic basis for the inactivity of this STAT1(K544A/E545A) mutant. Rather than failing to attract transcriptional coactivators, the STAT1(K544A/E545A) mutant has a subtle biophysical defect, which prevents accumulation of the activated protein on chromatin in vivo: the mutant has comparable K(d) with greatly increased k(off) for DNA binding. The increase in both on-rate and off-rate of DNA binding results in a substantially reduced residence time of STAT1(K544A/E545A) on STAT binding sites. We find a similar correlation between off-rate and transcriptional potency for STAT1(N460A), which bears a mutation in the DNA binding domain. These results yield insight into the rate of complex assembly involving STAT1 that leads to transcriptional stimulation.
信号转导和转录激活因子1（STAT1）转录因子由几个高度保守的结构域组成，除连接结构域外，每个结构域都已被赋予特定功能。我们之前鉴定了STAT1连接结构域中的一个突变体，在电泳迁移率分析中，该突变体使用标准探针时能正常结合DNA，但对干扰素γ无转录激活反应。我们现在报告这种STAT1（K544A/E545A）突变体无活性的机制基础。STAT1（K544A/E545A）突变体并非无法吸引转录共激活因子，而是存在一个细微的生物物理缺陷，该缺陷阻止了活化蛋白在体内染色质上的积累：该突变体与DNA结合的解离常数（K(d)）相当，但解离速率常数（k(off)）大幅增加。DNA结合的结合速率和解离速率的增加导致STAT1（K544A/E545A）在STAT结合位点上的停留时间大幅缩短。我们发现，在DNA结合结构域中存在突变的STAT1（N460A）的解离速率与转录活性之间也存在类似的相关性。这些结果有助于深入了解涉及STAT1的复合物组装速率，而这种组装会导致转录激活。
