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Mechanistic role of bent DNA in transcription from eukaryotic promoters

弯曲DNA在真核启动子转录中的机制作用

基本信息

批准号：
09680681
负责人：
OHYAMA Takashi
金额：
$ 1.92万
依托单位：
KONAN UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680681/
关键词：
bent DNA promoter transcriptional regulation chromatin nucleosome eukaryotic transcription 転写調節 DNA高次構造

项目摘要

Unusual curved DNA structures are sometimes reported to reside within genetic regions that regulate prokaryotic and eukaryotic transcription. Extensive data suggesting a role in prokaryotic transcription for the structure have accumulated over the last few years. However, little has been revealed as to the role of DNA curvature in eukaryotic transcription.Recent studies indicated that DNA curvatures can directly activate eukaryotic transcription. In order to elucidate that mechanism, using herpes simplex thymidine kinase (tk) promoter or human adenovirus type 2 (Ad2) E1A promoter, we investigated the effects of synthetic plane and space curves on the promoter activities. Double-stranded oligonucleotides having various three-dimensional architectures were designed, synthesized and ligated to each promoter. Furthermore, rotational orientation of each synthetic DNA segment relative to each promoter was altered by deleting several nucleotides from the region between them. Luciferase assay showed that a bent DNA having left-handed superhelical writhe could remarkably activate tk promoter. In the process, rotational orientation of the writhe relative to the promoter and the distance between them played a very important role. Results of in vitro and in vivo DNase I footprinting assays indicated that introduction of synthetic DNA curvatures into upstream region of the tk promoter influenced nucleosome positioning on the promoter. The left-handed superhelical writhe directed positions of nucleosomes to prevent inappropriate inhibitory histone-DNA contacts from occurring over the TATA box. It is strongly indicated that in eukaryotic gene transcription, a role of bent DNA structures is to modulate the organization of local chromatin structure.

不寻常的弯曲DNA结构有时被报道存在于调节原核和真核转录的遗传区域内。在过去的几年里，大量的数据表明该结构在原核转录中的作用。然而，DNA曲率在真核生物转录中的作用却很少被研究，最近的研究表明DNA曲率可以直接激活真核生物的转录。为了阐明这一机制，我们分别用单纯疱疹病毒胸苷激酶（tk）启动子和人腺病毒2型（Ad 2）E1 A启动子，研究了合成平面和空间曲线对启动子活性的影响。设计、合成具有各种三维结构的双链寡核苷酸并将其连接到每个启动子。此外，每个合成DNA片段相对于每个启动子的旋转方向通过从它们之间的区域删除几个核苷酸来改变。荧光素酶分析表明，具有左旋超螺旋扭曲的弯曲DNA能显著激活tk启动子。在这个过程中，螺旋相对于启动子的旋转方向以及螺旋与启动子之间的距离起着非常重要的作用。体外和体内DNA酶I足迹分析的结果表明，在tk启动子的上游区域中引入合成的DNA曲率影响核小体在启动子上的定位。左手超螺旋扭曲核小体的定向位置，以防止TATA盒上发生不适当的抑制性组蛋白-DNA接触。这有力地表明，在真核基因转录中，DNA弯曲结构的作用是调节局部染色质结构的组织。