Rega Institute for Medical Research, Department of Microbiology, Immunology and Transplantation, KU Leuven, 3000 Leuven, Belgium.
Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ 08854, USA.
STAR Protoc. 2021 Mar 31;2(2):100431. doi: 10.1016/j.xpro.2021.100431. eCollection 2021 Jun 18.
In yeast mitochondria, transcription initiation requires assembly of mitochondrial RNA polymerase and transcription initiation factor MTF1 at the DNA promoter initiation site. This protocol describes the purification of the component proteins and assembly of partially melted and fully melted initiation complex states. Both states co-exist in equilibrium in the same sample as seen by cryoelectron microscopy (cryo-EM) and allow elucidation of MTF1's structural roles in controlling the transition into elongation. We further outline how analysis of the complex by light scattering, thermal shift assay, and ultrafiltration assay exhibits reproducible results. For complete details on the use and execution of this protocol, please refer to De Wijngaert et al. (2021).
在酵母线粒体中，转录起始需要线粒体 RNA 聚合酶和转录起始因子 MTF1 在 DNA 启动子起始位点组装。本方案描述了组分蛋白的纯化以及部分融化和完全融化起始复合物状态的组装。如冷冻电子显微镜 (cryo-EM) 所示，这两种状态在同一样品中处于平衡共存状态，并允许阐明 MTF1 在控制进入延伸过程中的结构作用。我们进一步概述了如何通过光散射、热位移测定和超滤测定分析复合物，以获得可重复的结果。有关该方案使用和执行的完整详细信息，请参阅 De Wijngaert 等人（2021 年）。
