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 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.
All Science Journal Classification (ASJC) codes
- Biochemistry, Genetics and Molecular Biology(all)
- Immunology and Microbiology(all)
- Protein expression and purification
- Structural Biology