IDENTIFICATION OF A MINIMAL SET OF PROTEINS THAT IS SUFFICIENT FOR ACCURATE INITIATION OF TRANSCRIPTION BY RNA POLYMERASE-II
IDENTIFICATION OF A MINIMAL SET OF PROTEINS THAT IS SUFFICIENT FOR ACCURATE INITIATION OF TRANSCRIPTION BY RNA POLYMERASE-II
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DOI：
10.1101/gad.7.7a.1254
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发表时间：
1993-07-01
影响因子：
10.5
通讯作者：
KADONAGA, JT
中科院分区：
文献类型：
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作者：
TYREE, CM;GEORGE, CP;KADONAGA, JT
在真核生物中，mRNA合成的启动是由RNA聚合酶II和通常称为基础或一般因素的辅助因子进行的多步过程。在这项研究中，通过纯化的，大肠杆菌合成的TFIIB，TBP，TBP（TFIID复合物的TATA盒结合多肽）和TFIIF的30 kD亚基（也称为RAP30）以及纯化的RNA Polosops ii frosospare frosope ryfiif（也称为RAP30）和30 kD亚基。这组最小的因素能够转录所测试的启动子的子集。 TFIIE的两个亚基和TFIIF的74 kD亚基都提高了转录效率的效率2至4。相反，相比之下，包括果蝇胚胎的粗TFIID分数，代替了重组TBP，这导致了对TFIIE的强烈依赖。通过凝胶迁移率分析，TFIIB，TBP，RAP30和聚合酶能够与具有转录能力（野生型或启动突变体）的DB和DBPolf30复合物组装成DB和DBPolf30复合物，但并非具有转录性不活性（TATA和TATA/TATA/tata/stapitation），Drosophila Adh的版本。因此，似乎RNA聚合酶II能够在组装DBPOLF30复合物之后启动转录，该复合物是一种微型转录复合物，代表了RNA聚合酶II转录机械的中心核心。
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.