Structural and Functional Roles of Modules in Protein Architecture

球状蛋白质由紧密的模块组成。蛋白质的模块边界与编码蛋白质的基因中的内含子位置密切相关。这一事实表明基因外显子的洗牌是蛋白质水平上的模块洗牌，模块本身是原始酶。该项目的目的是获得模块作为原始酶的支持证据，并了解自然界中蛋白质设计的原理。本研究使用的蛋白质有细菌RNA酶——藤蔓酶、RNA聚合酶II、原核生物转录抑制因子和叶绿体中的核糖核蛋白等。将Barnase分解为6个模块（M1-M6）。氢键主要位于藤本酶的模组内。这一事实表明，氢键的形成有助于确定藤本酶结构中模块的紧密构象。我们化学合成了模块，并对每个模块在溶液中的构象及其功能进行了实验研究。三个模块（M2， M3和M6）被发现具有RNase的酶促功能。这是第一次报道模块的酶功能。我们的结果清楚地表明，这些模块在早期进化中起着原始酶的作用。我们还使用2D-NMR和距离几何确定了模块M2和M3的二级结构。它们的二级结构与完整的藤壶中发现的相似。此外，我们在RNA聚合酶II中发现了一个类似藤壶的结构域，并预测了该区域RNA聚合酶的催化功能。其他研究组报道了RNA聚合酶II的RNA酶活性。我们建议这个区域可能参与校对，如果有的话。通过对转录阻遏物的模块组织分析，我们发现一些阻遏物具有两个或三个螺旋-转-螺旋模块，并讨论了阻遏物通过模块组合的进化过程。

A globular protein consists of compact modules. Module boundaries of proteins are closely correlated with intron positions in the genes encoding the proteins. This fact implies that exon-shuffling in genes is module shuffling in protein level and modules themselves were primitive enzymes. The purpose of this project is to obtain supporting evidences of modules as primitive enzymes and to understand the principle of protein design in nature. Proteins used in this study were barnase that is a bacterial RNase, RNA polymerase II, transcription repressors of procaryotes and ribonuclear proteins in chloroplast, etc. Barnase was decomposed into six modules(M1-M6). Hydrogen bonds were located predominantly within modules in barnase. This fact shows that hydrogen bond formation contributes to determine the compact conformation of modules in barnase architecture. We synthesized modules chemically and carried out experimental study on conformation of each module in solution and its function. Three modules (M2, M3 and M6) were revealed to have an enzymatic function as RNase. This is the first report on the enzymatic function of modules. Our results clearly imply that the modules carried a role as primitive enzymes in early evolution. We also determined the secondary structures of modules M2 and M3 using 2D-NMR and distance geometry. They had the similar secondary structures to those found in intact barnase. Furthermore, we found a barnase-like domain in RNA polymerase II and predicted RNase catalytic function in this region. By other research groups RNase activity of RNA polymerase II was reported. We have suggested that this region might be involved in proof reading, if any. By analysis of module organization in transcription repressors, we found that some of them have two or three helix-turn-helix modules and discussed evolutionary process of repressors through module combinations.