Developmental research on the microscopical imaging of DNA transcription process.

本项目旨在建立DNA转录过程的成像方法，特别是DNA、RNA聚合酶和调控蛋白复合体的三维组装。为此，我们开发了第一台分子冷冻干燥装置，可用于原子力成像。然而，原子力显微镜不能描述DNA-蛋白质复合物的分子结构。相当经典的低角度阴影法明显地检测到了复杂的结构。为了提高阴影法的分辨率，我们研制了一种工作在极高真空（10 μ P）下的新型冷冻蚀刻机<-6>。在-100 ° C下蒸发的铂颗粒非常小，以至于DNA的基本结构以及DNA结合蛋白（如聚合酶）的分子组织可以通过低角度旋转阴影来描绘，因此，我们专门使用这种技术来成像DNA转录过程。高分辨率旋转阴影法显示大肠杆菌RNA聚合酶由5个亚基组成，这与以前的生化证据是一致的。根据亚基的分子量，分别鉴定了对应于α、β、γ亚基的结构。α，α和β，β '亚基形成两个通道。其中较大的一个位于2 α和β之间。另一个是在β和β之间发现的，就像一条缝。组装的DNA总是在2 α和β、β '之间的凹槽中观察到。这表明新合成的mRNA可能是沿着β和β '形成的缝隙沿着向上。

This project aimed to establish the imaging methods of transcription process of DNA,especially three dimensionalassembly of DNA,RNA polymerase and regulatory proteins complex. For this purpose, we developed first freeze-drying apparatus by molecular distilllation, that is available for atomic force imaging. Howefver, atomic force microscope was not able to described molecular architecture of DNA-protein complex. rather classical low angle shadowing detected obviously the complex structure. To improve the resolution of shadowing method, we developed new freeze etching machine working at extremely high vacuum (10^<-6> P). Platinum particles evaporated in this apparatus at-100ﾟC was so small that basic structure of DNA as well as molecular organization of DNA binding proteins such as polymerase was depicted by low angle rotary shadowing.therefore, we used this technique exclusively for imaging of DNA transcription processes. High resolution rotary shadowing revealed that E.coli RNA polymerase consisted of 5 subunits which is well consistentwith previous biochemical evidence. Depending upon molecular weight of subunits, structures corresponding to alpha, beta, gamma subunits were identified respectively. alpha, alpha andbeta, beta'subunit formed two channels. One of them, larger one, was located between 2 alphaandbeta, beta'. The another one was found between beta and beta'like a slit. Assembled DNA was always observed in the groove between 2alpha and beta, beta'. These are suggested that newlysynthesized mRNA may come up along the slit formed by betaandbeta'.