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

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.