Frontier Institute for Biomolecular Engineering Research (FIBER) , Konan University , 7-1-20 minatojima-Minamimachi , Kobe 650-0047 , Japan.
Graduate School of Frontiers of Innovative Research in Science and Technology (FIRST) , Konan University , 7-1-20 minatojima-Minamimachi , Kobe 650-0047 , Japan.
Biochemistry. 2019 Feb 26;58(8):1081-1093. doi: 10.1021/acs.biochem.8b01204. Epub 2019 Feb 15.
Since the origin of life on Earth, the role of carrying genetic information has been presumably transferred from RNA to DNA. At present, cellular environments are extremely dense, packed with cosolutes and macromolecules. Hence, the preference between RNA-dependent RNA and DNA polymerization may be affected by molecular crowding. In this study, we investigated both RNA-dependent RNA and DNA polymerizations by tC9Y polymerase ribozyme, T7 RNA polymerase (T7 RNAP), and Klenow fragment DNA polymerase (KF) under different molecular crowding conditions. Poly(ethylene glycol) (PEG) of various molecular weights was used as a crowding agent and found to promote both RNA and DNA ribozyme-catalyzed polymerizations. In contrast, PEG with an average molecular weight of 200 (PEG200) reduced the level of RNA polymerization by proteinaceous T7 RNAP but simultaneously promoted DNA polymerization, without affecting the activity of KF. Thus, proteinaceous RNA polymerase might potentially display bisubstrate specificity, which can be switched in response to changes in the dielectric constant and excluded volume in crowded environments. Our findings validate the bisubstrate activity of RNA polymerase from an evolutionary perspective for the development of non-natural materials.
自地球上生命起源以来，携带遗传信息的角色可能已经从 RNA 转移到了 DNA。目前，细胞环境极其密集，充满了共溶剂和大分子。因此，RNA 依赖性 RNA 和 DNA 聚合之间的偏好可能会受到分子拥挤的影响。在这项研究中，我们在不同的分子拥挤条件下，用 tC9Y 聚合酶核酶、T7 RNA 聚合酶 (T7 RNAP) 和 Klenow 片段 DNA 聚合酶 (KF) 研究了 RNA 依赖性 RNA 和 DNA 的聚合。使用各种分子量的聚乙二醇 (PEG) 作为拥挤剂，发现它促进了 RNA 和 DNA 核酶催化的聚合。相比之下，分子量平均为 200 的 PEG200 降低了蛋白质 T7 RNAP 的 RNA 聚合水平，但同时促进了 DNA 聚合，而不影响 KF 的活性。因此，蛋白质 RNA 聚合酶可能具有双底物特异性，可以根据拥挤环境中介电常数和排除体积的变化进行切换。我们的发现从进化的角度验证了 RNA 聚合酶的双底物活性，这对于开发非天然材料具有重要意义。
