Tyree C M, George C P, Lira-DeVito L M, Wampler S L, Dahmus M E, Zawel L, Kadonaga J T
Department of Biology and Center for Molecular Genetics, University of California, San Diego La Jolla 92093.
Genes Dev. 1993 Jul;7(7A):1254-65. doi: 10.1101/gad.7.7a.1254.
In eukaryotes, initiation of mRNA synthesis is a multistep process that is carried out by RNA polymerase II and auxiliary factors that are commonly referred to as basal or general factors. In this study accurate initiation of transcription was reconstituted with purified, Escherichia coli-synthesized TFIIB, TBP (the TATA box-binding polypeptide of the TFIID complex), and the 30-kD subunit of TFIIF (also known as RAP30), along with purified, native RNA polymerase II from Drosophila embryos, calf thymus, or HeLa cells. This minimal set of factors was able to transcribe a subset of the promoters tested. The addition of both subunits of TFIIE and the 74-kD subunit of TFIIF increased the efficiency of transcription by a factor of 2 to 4. In contrast, the inclusion of a crude TFIID fraction from Drosophila embryos in place of recombinant TBP resulted in a strong dependence on TFIIE. By gel mobility-shift analysis, TFIIB, TBP, RAP30, and polymerase were able to assemble into DB and DBPolF30 complexes with transcriptionally competent (wild type or initiator mutant), but not with transcriptionally inactive (TATA and TATA/initiator mutant), versions of the Drosophila Adh promoter. Thus, it appears that RNA polymerase II is able to initiate transcription subsequent to assembly of the DBPolF30 complex, which is a minitranscription complex that represents the central core of the RNA polymerase II transcriptional machinery.
在真核生物中，mRNA合成的起始是一个多步骤过程，由RNA聚合酶II和通常被称为基础或通用因子的辅助因子来完成。在本研究中，利用纯化的、大肠杆菌合成的TFIIB、TBP（TFIID复合物的TATA盒结合多肽）和TFIIF的30-kD亚基（也称为RAP30），以及从果蝇胚胎、小牛胸腺或HeLa细胞中纯化的天然RNA聚合酶II，成功重构了准确的转录起始过程。这一最小的因子组合能够转录所测试启动子的一个子集。添加TFIIE的两个亚基和TFIIF的74-kD亚基可使转录效率提高2至4倍。相比之下，用果蝇胚胎的粗制TFIID组分替代重组TBP会导致对TFIIE的强烈依赖。通过凝胶迁移率变动分析，TFIIB、TBP、RAP30和聚合酶能够与具有转录活性（野生型或起始子突变体）的果蝇Adh启动子版本组装成DB和DBPolF³⁰复合物，但不能与转录无活性（TATA和TATA/起始子突变体）的版本组装。因此，似乎RNA聚合酶II能够在DBPolF³⁰复合物组装后起始转录，DBPolF³⁰复合物是一种微型转录复合物，代表了RNA聚合酶II转录机制的核心。
