Work

Protocol to investigate human mitochondrial transcription initiation by integrating biochemical and cryo-EM approaches

Year: 2026

Type: article

Abstract: Here, we present a protocol to investigate the mechanism and regulation of human mitochondrial transcription initiation by integrating biochemical and cryoelectron microscopy (cryo-EM) approaches. We describe the steps for expressing and purifying POLRMT, TFB2M, and TFAM; assembling transcription initiation complexes; and utilizing single-particle cryo-EM analysis for the high-resolution structural determination of transcription intermediates. We provide assays for quantifying promoter melting, ATP binding, abortive RNA synthesis, and RNA slippage steps, enabling structure-function studies and gaining mechanistic insights into transcription initiation and transcription factor regulation. For complete details on the use and execution of this protocol, please refer to Shen et al. 1 • Protocols for purification of POLRMT, TFB2M, and TFAM proteins • Biochemical assays to analyze transcription initiation and RNA slippage • Technique for assembling human mitochondrial transcription initiation complexes • Procedure for cryo-EM grid preparation, data collection, and structure determination Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol to investigate the mechanism and regulation of human mitochondrial transcription initiation by integrating biochemical and cryoelectron microscopy (cryo-EM) approaches. We describe the steps for expressing and purifying POLRMT, TFB2M, and TFAM; assembling transcription initiation complexes; and utilizing single-particle cryo-EM analysis for the high-resolution structural determination of transcription intermediates. We provide assays for quantifying promoter melting, ATP binding, abortive RNA synthesis, and RNA slippage steps, enabling structure-function studies and gaining mechanistic insights into transcription initiation and transcription factor regulation.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.104347

Authors: Yogeeshwar Ajjugal, Quinten Goovaerts, Jiayu Shen, Brent De Wijngaert, Kalyan Das, Smita S. Patel

Institutions: Rutgers, The State University of New Jersey, Rega Institute for Medical Research, KU Leuven, University of Alberta

Language: English

Topic: Genomics and Chromatin Dynamics

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $1,550