A MEDIATOR REQUIRED FOR ACTIVATION OF RNA POLYMERASE-II TRANSCRIPTION INVITRO
A MEDIATOR REQUIRED FOR ACTIVATION OF RNA POLYMERASE-II TRANSCRIPTION INVITRO
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DOI：
10.1038/350436a0
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发表时间：
1991-04-04
期刊：
影响因子：
64.8
通讯作者：
KORNBERG, RD
中科院分区：
文献类型：
--
作者：
FLANAGAN, PM;KELLEHER, RJ;KORNBERG, RD
激活蛋白与增强子DNA元件结合并刺激转录的启动。 已经提出1-3，激活剂接触启动子处的一般起始因子，并且已经获得了这种直接相互作用的证据4-10。 然而，在体外转录的研究表明，激活剂可能通过与一般因素不同的中间分子（S）发挥作用。 在这些研究中的第一个11,12中，我们利用了一个发现，即一种激活物可以抑制第二个激活剂（激活剂干扰或“挤压”）13-15刺激的转录。 这种抑制作用归因于激活剂之间对共同目标因子的竞争，因此无法通过添加大量过量的一般启动因子来缓解，这表明激活者竞争的目标与这些因素不同。 类似的结论也是从克隆基因16-18表达的TFIID未能替换在支持激活时部分纯化的“天然” TFIID馏分的观察结果，这显然是因为它们在不纯净的部分中缺乏某些组件。 虽然这些新型“介体”激活的证据是负面的，但我们还表明，酵母中部分纯化的部分会逆转激活剂的干扰12。推定介体的这种积极作用为其活性提供了一种测定，但其在其在其中的作用，但它在激活仍然仅推断。 现在，我们提供了介体的直接证据，这是激活剂GAL4-VP16和GCN4在体外刺激转录所必需的，但在没有激活蛋白的情况下对转录没有影响。
ACTIVATOR proteins bind to enhancer DNA elements and stimulate the initiation of transcription. It has been proposed 1-3 that activators contact general initiation factors at a promoter, and evidence for such direct interaction has been obtained 4-10. Studies of transcription in vitro, however, have suggested that activators might function through an intermediary molecule(s) distinct from the general factors. In the first of these studies 11,12, we exploited the finding that one activator could inhibit transcription stimulated by a second activator (activator interference or 'squelching') 13-15. This inhibition, which is attributed to competition between the activators for a common target factor, could not be relieved by addition of a large excess of general initiation factors, suggesting that the target for which activators compete is distinct from these factors. Similar conclusions came from the observation that TFIID's expressed from cloned genes 16-18 fail to replace partially purified 'natural' TFIID fractions in supporting activation, evidently because they lacked some component present in the impure fractions. While these lines of evidence for a novel 'mediator' of activation were negative, we also showed that a partially purified fraction from yeast would reverse activator interference 12. This positive effect of a presumptive mediator provided an assay for its activity, but its role in activation was still only inferred. We now present direct evidence for a mediator which is required for stimulation of transcription in vitro by the activators GAL4-VP16 and GCN4, but which has no effect on transcription in the absence of activator protein.