Assembly and Cryo-EM structure determination of yeast mitochondrial RNA polymerase initiation complex intermediates

被引：2

作者：

Martinez, Sergio E. ^{[1
]}

Singh, Anupam ^{[2
]}

De Wijngaert, Brent ^{[1
]}

Sultana, Shemaila ^{[2
]}

Dharia, Chhaya ^{[2
]}

Vanbuel, Hans ^{[1
]}

Shen, Jiayu ^{[2
]}

Vasilchuk, Daniel ^{[2
]}

Patel, Smita S. ^{[2
]}

Das, Kalyan ^{[1
]}

机构：

[1] Katholieke Univ Leuven, Rega Inst Med Res, Dept Microbiol Immunol & Transplantat, B-3000 Leuven, Belgium

[2] Rutgers State Univ, Robert Wood Johnson Med Sch, Dept Biochem & Mol Biol, Piscataway, NJ 08854 USA

来源：

STAR PROTOCOLS | 2021年 / 2卷 / 02期

关键词：

WILD-TYPE;

D O I：

10.1016/j.xpro.2021.100431

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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 micro-scopy (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 repro-ducible results. For complete details on the use and execution of this protocol, please refer to De Wijngaert et al. (2021).

引用

页数：15

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