Specific 5' flanking sequences are required for faithful initiation of in vitro transcription of the ovalbumin gene.
Specific 5' flanking sequences are required for faithful initiation of in vitro transcription of the ovalbumin gene.
复制标题
卵清蛋白基因体外转录的忠实起始需要特定的 5 侧翼序列。
DOI：
10.1073/pnas.78.2.879
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发表时间：
1981
影响因子：
11.1
通讯作者：
O'Malley,BW
中科院分区：
文献类型：
--
作者：
Tsai,SY;Tsai,MJ;O'Malley,BW
Anin vitrosystem [Weil，P.A.，Luse，D.美国，Segall，J. & Roeder，R. G.（1979）Cell 18，469-484和Manley，J.L.，火，A.，卡诺，A.，夏普，宾夕法尼亚州。& Gefter，M. L.，等人（1980）Proc. Acad. Sci USA 77，3855-3859]适用于研究卵清蛋白基因转录的起始。模板DNA是克隆的卵清蛋白基因片段，其包含5′侧翼序列、第一结构序列区和第一间插序列的一部分。使用HeLa细胞粗提物作为RNA聚合酶和起始因子的来源。通过在基因3′端之前的不同位置截短卵清蛋白模板产生的转录产物的大小来判断正确的起始。根据α-鹅膏蕈碱敏感性判断，通过RNA聚合酶II进行特异性产物的转录。利用核酸外切酶III对卵清蛋白DNA模板的5′侧翼序列进行剪切，构建了一系列缺失突变体。然后将产生的DNA克隆到pBR 322中并用作模板以确定哪些序列是转录起始所必需的。卵清蛋白基因的特异性起始不受5′侧翼序列中除61个核苷酸外的所有核苷酸的缺失的影响，但完全被5′侧翼序列中除26个核苷酸外的所有核苷酸的缺失所消除。因此，在帽位点上游61和26个核苷酸之间的区域，包括在32-26位的Hogness盒（T-A-T-A-T-A-T），对于卵清蛋白基因的正确起始是必需的。然而，含有假Hogness盒（T-A-T-A-A-A-A和T-A-T-A-T-A-T）的天然DNA片段通常不位于真实基因的5 '侧翼区域，并不能作为转录起始的启动子。这些结果表明，Hogness框是必不可少的，但不足以特异性启动RNA合成。
Anin vitrosystem [Weil, P. A., Luse, D. S., Segall, J. & Roeder, R. G. (1979)Cell18, 469-484 and Manley, J. L., Fire, A., Cano, A., Sharp, P. A. & Gefter, M. L., (1980)Proc. Natl. Acad. Sci USA77, 3855-3859] was adapted for studying initiation of transcription of the ovalbumin gene. The DNA template was a cloned ovalbumin gene fragment that contained 5′ flanking sequences, the first structural sequence region, and a portion of the first intervening sequence. A HeLa cell crude extract was used as the source of RNA polymerase and initiation factors. Correct initiation was judged by the sizes of the transcription products generated from ovalbumin templates truncated at various positions before the 3′ end of the gene. Transcription of the specific product was carried out by RNA polymerase II, as judged from α-amanitin sensitivity. A series of deletion mutants was constructed by trimming 5′ flanking sequences of the ovalbumin DNA template by using exonuclease III. The DNAs generated were then cloned in pBR322 and used as templates to determine which sequences were necessary for initiation of transcription. Specific initiation of the ovalbumin gene was unaffected by deletion of all but 61 nucleotides of the 5′ flanking sequence but completely abolished by deletion of all but 26 nucleotides of 5′ flanking sequence. Thus, a region between 61 and 26 nucleotides upstream from the cap site, which includes the Hogness box (T-A-T-A-T-A-T) at position 32-26, is essential for the correct initiation of the ovalbumin gene. Nevertheless, natural DNA fragments containing false Hogness boxes (T-A-T-A-A-A-A and T-A-T-A-T-A-T) not normally located in the immediate 5′ flanking region of an authentic gene did not serve as promoters for initiation of transcription. These results suggest that the Hogness box is essential, but not sufficient, for specific initiation of RNA synthesis.