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

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).

Keywords: Cryo-EM; Protein expression and purification; Structural Biology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryoelectron Microscopy / methods*
  • DNA-Directed RNA Polymerases* / chemistry
  • DNA-Directed RNA Polymerases* / metabolism
  • DNA-Directed RNA Polymerases* / ultrastructure
  • Mitochondrial Proteins* / chemistry
  • Mitochondrial Proteins* / metabolism
  • Mitochondrial Proteins* / ultrastructure
  • Mitochondrial Ribosomes
  • Saccharomyces cerevisiae Proteins* / chemistry
  • Saccharomyces cerevisiae Proteins* / metabolism
  • Saccharomyces cerevisiae Proteins* / ultrastructure
  • Transcription Factors* / chemistry
  • Transcription Factors* / metabolism
  • Transcription Factors* / ultrastructure


  • MTF1 protein, S cerevisiae
  • Mitochondrial Proteins
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • DNA-Directed RNA Polymerases