Saccharomyces cerevisiae Med9 comprises two functionally distinct domains that play different roles in transcriptional regulation
Saccharomyces cerevisiae Med9 comprises two functionally distinct domains that play different roles in transcriptional regulation
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DOI：
10.1111/j.1365-2443.2008.01250.x
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发表时间：
2009-01
期刊：
影响因子：
2.1
通讯作者：
Hiroyuki Takahashi;K. Kasahara;T. Kokubo
中科院分区：
文献类型：
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作者：
Hiroyuki Takahashi;K. Kasahara;T. Kokubo
中介体是真核生物转录调控中最重要的共激活因子之一。在酿酒酵母（Saccharomyces cerevisiae）中，Mediator由25个亚基组成，属于4个结构不同的模块：Head， Middle， Tail和Cyc‐C。尽管每个模块在一组不同基因的调控中起着关键作用，但其精确的分子机制尚不清楚。为了进一步了解较少表征的Middle模块的作用，我们通过构建一组突变体并对其进行一系列体内和体外实验来分析Med9的功能。我们的研究结果表明Med9具有两个功能域。物种特异性氨基末端一半（aa 1-63）在体内和体外的转录调控中起调节作用。相比之下，保守的羧基末端部分（aa 64-149）具有更基本的功能，涉及直接结合Med4和Med7的氨基末端部分以及Med9组装到Middle模块中。重要的是，激活剂依赖的TBP和Taf11对启动子的募集在med9提取物和缺乏Mediator的提取物中受到不同的影响。加回实验表明，med9提取物中的一些未知因子可能会影响TFIID与启动子的结合。
Mediator is one of the most important co‐activators that function in eukaryotic transcriptional regulation. In Saccharomyces cerevisiae, Mediator is comprised of 25 subunits belonging to four structurally distinct modules: Head, Middle, Tail, and Cyc‐C. Although each module plays a critical role in the regulation of a distinct set of genes, the precise molecular mechanisms remain unclear. To gain new insight into the role of the less‐characterized Middle module, we analyzed the function of Med9 by constructing a set of mutants and subjecting them to a range of in vivo and in vitro assays. Our results demonstrate that Med9 has two functional domains. The species‐specific amino‐terminal half (aa 1–63) plays a regulatory role in transcriptional regulation in vivo and in vitro. In contrast, the well‐conserved carboxy‐terminal half (aa 64–149) has a more fundamental function involved in direct binding to the amino‐terminal portions of Med4 and Med7 and the assembly of Med9 into the Middle module. Importantly, activator‐dependent recruitment of TBP and Taf11 to the promoter is differentially affected in med9 extracts and in extracts lacking Mediator. Add‐back experiments indicate that some unidentified factor(s) in med9 extracts may impact the binding of TFIID to the promoter.