Fedorova Olga
Howard Hughes Medical Institute and Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA.
Methods Mol Biol. 2012;848:91-111. doi: 10.1007/978-1-61779-545-9_7.
Group II introns are large self-splicing ribozymes found in bacterial genomes, in organelles of plants and fungi, and even in some animal organisms. Many organellar group II introns interrupt important housekeeping genes; therefore, their splicing is critical for the survival of the host organism. Group II introns are versatile catalytic RNAs: they facilitate their own excision from a pre-mRNA, they promote ligation of exons to form a translation-competent mature mRNA; they can act like mobile genomic elements and insert themselves into RNA and DNA targets with remarkable precision, which makes them attractive tools for genetic engineering. The first step in characterization of any group II intron is the evaluation of its catalytic activity and its ability to properly fold into the native functionally active structure. This chapter describes kinetic assays used to characterize folding and catalytic properties of group II intron-derived ribozymes.
II 类内含子是在细菌基因组、植物和真菌的细胞器甚至一些动物生物体中发现的大型自我剪接核酶。许多细胞器 II 类内含子会中断重要的管家基因；因此，它们的剪接对于宿主生物体的生存至关重要。II 类内含子是多功能催化 RNA：它们促进自身从前体 mRNA 中切除，促进外显子连接以形成具有翻译能力的成熟 mRNA；它们可以像移动基因组元件一样发挥作用，并以极高的精度将自身插入 RNA 和 DNA 靶标，这使其成为基因工程中有吸引力的工具。表征任何 II 类内含子的第一步是评估其催化活性以及正确折叠成天然功能活性结构的能力。本章描述了用于表征 II 类内含子衍生核酶的折叠和催化特性的动力学测定方法。
