Jones G, Manczak M, Schelling D, Turner H, Jones D
School of Biological Sciences, Molecular and Cellular Biology Section, University of Kentucky, Lexington, Kentucky 40506, USA.
Biochem J. 1998 Oct 1;335 ( Pt 1)(Pt 1):79-84. doi: 10.1042/bj3350079.
The binding of transcription factors to the core promoter of the juvenile hormone esterase gene was functionally characterized using both a cell-free in vitro transcription functional assay and a cell transfection assay. A core JHE promoter (-61 to +28 bp relative to transcription start site) supported faithful transcription from the in vivo transcription start site. The nuclear extracts from the Sf9 insect cell line that provided transcription from that template also bound to that template as a probe in gel-mobility shift assays. Deletion or transversion of the initiator-binding motif (-1 to +4 bp) abolished detectable transcription either in vitro or in transfected cells. An AT-rich motif (ATATAT; -28 to -23 bp) serves another transcription factor-binding site. Mutation of the AT-rich motif to a canonical TATA-box preserved transcription, while either its deletion or complete transversion abolished or significantly reduced detectable transcriptional activity. These results indicate that, under these conditions, the functional operation of this core promoter approaches that of a composite promoter in which both the TATA- and initiator-binding protein complexes are necessary, even for basal transcription. On the other hand, these debilitating mutations to either the TATA box or initiator motif did not prevent the ability of the corresponding gel-shift competitive probes to compete with the wild-type promoter for binding by the transcription factors. Even a double transversion of both the AT-rich motif and the initiator-binding motif was able to competitively displace the protein complex that bound to the labelled wild-type probe. These data strongly indicate the presence of (an) additional core-promoter-associated transcription factor(s) (that is not the 'downstream element') that contact(s) the AT-binding complex and/or initiator-binding factor with sufficient avidity to remove them from binding to the competing wild-type promoter sequence.
利用无细胞体外转录功能测定法和细胞转染测定法，对转录因子与保幼激素酯酶基因核心启动子的结合进行了功能表征。一个核心JHE启动子（相对于转录起始位点为-61至+28 bp）支持从体内转录起始位点进行准确转录。来自Sf9昆虫细胞系的核提取物能从该模板进行转录，在凝胶迁移率变动分析中，该核提取物也能作为探针与该模板结合。起始子结合基序（-1至+4 bp）的缺失或颠换消除了体外或转染细胞中可检测到的转录。一个富含AT的基序（ATATAT；-28至-23 bp）是另一个转录因子结合位点。将富含AT的基序突变为典型的TATA框可保留转录，而其缺失或完全颠换则消除或显著降低了可检测到的转录活性。这些结果表明，在这些条件下，该核心启动子的功能运作类似于复合启动子，其中TATA结合蛋白复合物和起始子结合蛋白复合物对于基础转录都是必需的。另一方面，这些对TATA框或起始子基序的削弱性突变并未阻止相应凝胶迁移竞争探针与野生型启动子竞争转录因子结合的能力。即使富含AT的基序和起始子结合基序都发生双颠换，也能够竞争性地取代与标记的野生型探针结合的蛋白质复合物。这些数据有力地表明存在（一种）额外的与核心启动子相关的转录因子（不是“下游元件”），它以足够的亲和力与AT结合复合物和/或起始子结合因子接触，从而使其无法与竞争性野生型启动子序列结合。
