Kaufmann J, Verrijzer C P, Shao J, Smale S T
Howard Hughes Medical Institute, University of California, Los Angeles School of Medicine 90095-1662, USA.
Genes Dev. 1996 Apr 1;10(7):873-86. doi: 10.1101/gad.10.7.873.
The core promoters for mammalian protein-coding genes often contain a TATA box, an initiator (Inr) element, or both of these control elements. The TFIID complex is essential both for TATA activity and for the activity of a common class of Inr elements characterized by an approximate consensus sequence PyPyA+1NT/APyPy. Although the complete set of proteins required for basal TATA-mediated transcription has been established, the requirements for TFIID-dependent Inr activity remain undefined. In this study we set out to reconstitute Inr activity with purified and recombinant general transcription factors. For this analysis, Inr activity was measured as the ability of an Inr to enhance the strength of a core promoter containing an upstream TATA box. Inr activity was not detected in reactions containing TFIIB, RAP30, RAP74, RNA polymerase II, and either TBP or TFIID, even though these factors were sufficient for TATA-mediated transcription from supercoiled templates. By use of a complementation assay, a factor that imparts Inr activity was identified. This factor, named CIF, stimulated Inr activity in reactions containing the TFIID complex, but activity was not detected with TBP. Further characterization of CIF suggested that it contains multiple components. Functional and immunological experiments demonstrated that one of the CIF components is the mammalian homolog of Drosophila TAF(II)150, which is not tightly associated with mammalian TFIID. These results reveal significant differences in the factor requirements for basal TATA and Inr activity. Further elucidation of these differences is likely to explain the need for the core promoter heterogeneity found within protein-coding genes.
哺乳动物蛋白质编码基因的核心启动子通常包含一个TATA框、一个起始子（Inr）元件，或这两种控制元件。TFIID复合物对于TATA活性以及一类以近似共有序列PyPyA+1NT/APyPy为特征的常见Inr元件的活性均至关重要。尽管已经确定了基础TATA介导转录所需的完整蛋白质组，但TFIID依赖性Inr活性的要求仍不明确。在本研究中，我们着手用纯化的和重组的通用转录因子重建Inr活性。对于此分析，Inr活性通过Inr增强含有上游TATA框的核心启动子强度的能力来衡量。在含有TFIIB、RAP30、RAP74、RNA聚合酶II以及TBP或TFIID的反应中未检测到Inr活性，尽管这些因子足以从超螺旋模板进行TATA介导的转录。通过使用互补分析，鉴定出一种赋予Inr活性的因子。这个名为CIF的因子在含有TFIID复合物的反应中刺激Inr活性，但用TBP未检测到活性。对CIF的进一步表征表明它包含多个组分。功能和免疫实验证明，CIF的一个组分是果蝇TAF(II)150的哺乳动物同源物，它与哺乳动物TFIID没有紧密关联。这些结果揭示了基础TATA和Inr活性在因子需求上的显著差异。对这些差异的进一步阐明可能解释了蛋白质编码基因中核心启动子异质性的必要性。
