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

Sergio E. Martinez, Anupam Singh, Brent De Wijngaert, Shemaila Sultana, Chhaya Dharia, Hans Vanbuel, Jiayu Shen, Daniel Vasilchuk, Smita S. Patel, Kalyan Das

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Article number100431
JournalSTAR Protocols
Volume2
Issue number2
DOIs
StatePublished - Jun 18 2021

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Neuroscience(all)
  • Immunology and Microbiology(all)

Keywords

  • Cryo-EM
  • Protein expression and purification
  • Structural Biology

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