Walker S, Greaves R, O'Hare P
Marie Curie Research Institute, The Chart, Oxted, Surrey, United Kingdom.
Mol Cell Biol. 1993 Sep;13(9):5233-44. doi: 10.1128/mcb.13.9.5233-5244.1993.
In this work we have examined the requirements for activity of the acidic domain of Vmw65 (VP16) by deletion and site-directed mutagenesis of the region in the context of GAL4 fusion proteins. The results indicate that the present interpretation of what actually constitutes the activation domain is not correct. We demonstrate, using a promoter with one target site which is efficiently activated by the wild-type (wt) fusion protein, that amino acids distal to residue 453 are critical for activity. Truncation of the domain or substitution of residues in the distal region almost completely abrogate activity. However, inactivating mutations within the distal region are complemented by using a promoter containing multiple target sites. Moreover, duplication of the proximal region, but not the distal region, restores the ability to activate a promoter with a single target site. These results indicate some distinct qualitative difference between the proximal and distal regions. We have also examined the binding of nuclear proteins to the wt domain and to a variant with the distal region inactivated by mutation. The lack of activity of this variant is not explained by a lack of binding of TFIIB, a protein previously reported to be the likely target of the acidic domain. Therefore some additional function is involved in transcriptional activation by the acid domain, and determinants distinct from those involved in TFIIB binding are required for this function. Analysis of the total protein profiles binding to the wt and mutant domains has demonstrated the selective binding to the wt domain of a 135-kDa polypeptide, which is therefore a candidate component involved in this additional function. This is the first report to provide evidence for the proposal of a multiplicity of interactions within the acidic domain, by uncoupling requirements for one function from those for another.
在这项研究中，我们通过在GAL4融合蛋白背景下对该区域进行缺失和定点诱变，研究了Vmw65（VP16）酸性结构域的活性要求。结果表明，目前对实际构成激活结构域的解释是不正确的。我们使用一个具有一个靶位点的启动子，该启动子能被野生型（wt）融合蛋白有效激活，结果表明453位残基远端的氨基酸对活性至关重要。该结构域的截短或远端区域残基的替换几乎完全消除了活性。然而，通过使用含有多个靶位点的启动子，可以弥补远端区域内的失活突变。此外，近端区域的重复，而不是远端区域的重复，恢复了激活具有单个靶位点的启动子的能力。这些结果表明近端和远端区域之间存在一些明显的质的差异。我们还研究了核蛋白与wt结构域以及远端区域因突变而失活的变体的结合情况。TFIIB是一种先前报道的可能是酸性结构域靶标的蛋白质，该变体缺乏活性并不能通过TFIIB结合的缺乏来解释。因此，酸性结构域的转录激活涉及一些额外的功能，并且该功能需要与参与TFIIB结合的决定因素不同的决定因素。对与wt和突变结构域结合的总蛋白谱的分析表明，一种135 kDa的多肽选择性地与wt结构域结合，因此它是参与这种额外功能的候选成分。这是第一份通过将一种功能的要求与另一种功能的要求解耦，为酸性结构域内存在多种相互作用的提议提供证据的报告。
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