IDENTIFICATION AND CHARACTERIZATION OF A TFIID-LIKE MULTIPROTEIN COMPLEX FROM SACCHAROMYCES-CEREVISIAE
IDENTIFICATION AND CHARACTERIZATION OF A TFIID-LIKE MULTIPROTEIN COMPLEX FROM SACCHAROMYCES-CEREVISIAE
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DOI：
10.1073/pnas.92.18.8224
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发表时间：
1995-08-29
影响因子：
11.1
通讯作者：
WEIL, PA
中科院分区：
文献类型：
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作者：
POON, D;BAI, Y;WEIL, PA
虽然RNA聚合酶II的转录调控机制显然是高度保守的从酵母到人，酵母TATA结合蛋白（TBP）-TBP相关因子（TAF（II））复合物的鉴定相当的后生动物TFIID组成部分的基础转录机器仍然难以捉摸。在这里，我们报告的分离酵母TBP-TAF（II）复合物，它可以介导转录激活的GAL 4-VP 16在一个高度纯化的酵母体外转录系统，我们已经克隆和测序的基因编码的四个多酵母TAF（II）蛋白组成的TBP-TAF（II）多亚基复合物，并发现它们是相似的，在氨基酸水平的人类和果蝇TFIID亚基。使用表位标记和免疫沉淀实验，我们证明了这些基因编码真正的TAF蛋白，并表明酵母TBP-TAF（II）复合物最低限度地由TBP和7个不同的yTAF（II）蛋白组成，大小从M（r）= 150，000到M（r）= 25，000。此外，通过构建空等位基因的克隆TAF编码基因，我们表明，正常功能的TAF编码基因是必不可少的酵母细胞的活力。
Although the mechanisms of transcriptional regulation by RNA polymerase II are apparently highly conserved from yeast to man, the identification of a yeast TATA-binding protein (TBP)-TBP-associated factor (TAF(II)) complex comparable to the metazoan TFIID component of the basal transcriptional machinery has remained elusive. Here, we report the isolation of a yeast TBP-TAF(II) complex which can mediate transcriptional activation by GAL4-VP16 in a highly purified yeast in vitro transcription system, We have cloned and sequenced the genes encoding four of the multiple yeast TAF(II) proteins comprising the TBP-TAF(II) multisubunit complex and find that they are similar at the amino acid level to both human and Drosophila TFIID subunits. Using epitope-tagging and immunoprecipitation experiments, we demonstrate that these genes encode bona fide TAF proteins and show that the yeast TBP-TAF(II) complex is minimally composed of TBP and seven distinct yTAF(II) proteins ranging in size from M(r) = 150,000 to M(r) = 25,000. In addition, by constructing null alleles of the cloned TAF-encoding genes, we show that normal function of the TAF-encoding genes is essential for yeast cell viability.