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.

有时据报道，异常的弯曲DNA结构驻留在调节原核生物和真核转录的遗传区域中。大量数据表明在过去几年中积累了该结构的原用转录作用。然而，关于DNA曲率在真核转录中的作用的揭示很少。截止性研究表明，DNA曲率可以直接激活真核转录。为了阐明该机制，使用疱疹单纯胸腺胺激酶（TK）启动子或人类腺病毒2型（AD2）E1A启动子，我们研究了合成平面和太空曲线对启动子活动的影响。设计，合成并连接到每个启动子的双链寡核苷酸。此外，通过删除它们之间区域的几个核苷酸，可以改变每个合成DNA段相对于每个启动子的旋转方向。荧光素酶测定法表明，具有左手超螺旋旋转的弯曲DNA可以显着激活TK启动子。在此过程中，旋转相对于启动子的旋转方向及其之间的距离起着非常重要的作用。体外和体内DNase I足迹测定的结果表明，将合成DNA曲率引入TK启动子的上游区域会影响启动子上的核小体定位。左撇子超螺旋旋转的定向核小体位置，以防止不适当的抑制性组蛋白-DNA接触发生在塔塔盒上。强烈表明，在真核基因转录中，弯曲DNA结构的作用是调节局部染色质结构的组织。