TATA-binding protein mutants that increase transcription from enhancerless and repressed promoters in vivo
TATA-binding protein mutants that increase transcription from enhancerless and repressed promoters in vivo
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DOI：
10.1128/mcb.20.5.1478-1488.2000
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发表时间：
2000-03-01
影响因子：
5.3
通讯作者：
Struhl, K
中科院分区：
文献类型：
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作者：
Geisberg, JV;Struhl, K
使用遗传筛选，我们分离出三个TATA结合蛋白（TBP）的突变体，增加从启动子的转录被抑制的Cyc 8-Tup 1或Sin 3-Rpd 3辅阻遏物或缺乏增强子元件，但不是从一个等效的弱启动子与突变的TATA元件。当TBP突变体在野生型TBP存在下以低水平表达时，观察到转录增加。这些TBP突变体无法支持细胞活力，并且在缺乏Rpd 3组蛋白脱乙酰酶的菌株中或在较高水平表达时具有毒性。虽然这些突变体在体外不能检测到与TATA元件结合，但遗传和染色质免疫沉淀实验表明，它们直接作用于启动子，并且不通过滴定负调节因子来增加转录。TBP突变体在与响应中等或强激活剂的启动子结合方面有轻度缺陷;此外，它们严重缺陷RNA聚合酶（Pol）III，但不缺陷Pol I转录。这些结果表明，相对于Pol II转录，TBP突变体特异性地增加核心启动子的表达。生化分析表明，TBP突变体不受TFIID复合物形成、二聚化以及与一般负调节因子NC 2或TAF 130的N-末端抑制结构域的相互作用的影响。我们推测这些TBP突变体具有不寻常的结构，使它们能够优先接近染色质模板中的TATA元件，这些TBP突变体定义了一个标准，通过该标准，被Cyc 8-Tup 1或Sin 3-Rpd 3阻遏的启动子类似于无增强子的启动子，但不是TATA缺陷的启动子;因此，他们支持这样的观点，即这些辅阻遏物抑制激活蛋白的功能，而不是Poi II机制。
Using a genetic screen, we isolated three TATA-binding protein (TBP) mutants that increase transcription from promoters that are repressed by the Cyc8-Tup1 or Sin3-Rpd3 corepressors or that lack an enhancer element, but not from an equivalently weak promoter with a mutated TATA element. Increased transcription is observed when the TBP mutants are expressed at low levels in the presence of wild-type TBP. These TBP mutants are unable to support cell viability, and they are toxic in strains lacking Rpd3 histone deacetylase or when expressed at higher levels. Although these mutants do not detectably bind TATA elements in vitro, genetic and chromatin immunoprecipitation experiments indicate that they act directly at promoters and do not increase transcription by titration of a negative regulatory factor(s), The TBP mutants are mildly defective for associating with promoters responding to moderate or strong activators; in addition, they are severely defective for RNA polymerase (Pol) III but not Pol I transcription. These results suggest that, with respect to Pol II transcription, the TBP mutants specifically increase expression from core promoters. Biochemical analysis indicates that the TBP mutants are unaffected for TFIID complex formation, dimerization, and interactions with either the general negative regulator NC2 or the N-terminal inhibitory domain of TAF130, We speculate that these TBP mutants have an unusual structure that allows them to preferentially access TATA elements in chromatin templates, These TBP mutants define a criterion by which promoters repressed by Cyc8-Tup1 or Sin3-Rpd3 resemble enhancerless, but not TATA-defective, promoters; hence, they support the idea that these corepressors inhibit the function of activator proteins rather than the Poi II machinery.