RESUMEN
Mitochondrial RNA polymerase (mtRNAP) is crucial in cellular energy production, yet understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. We report structures of two transcription initiation intermediate states of yeast mtRNAP that explain promoter melting, template alignment, DNA scrunching, abortive synthesis, and transition into elongation. In the partially melted initiation complex (PmIC), transcription factor MTF1 makes base-specific interactions with flipped non-template (NT) nucleotides "AAGT" at -4 to -1 positions of the DNA promoter. In the initiation complex (IC), the template in the expanded 7-mer bubble positions the RNA and NTP analog UTPαS, while NT scrunches into an NT loop. The scrunched NT loop is stabilized by the centrally positioned MTF1 C-tail. The IC and PmIC states coexist in solution, revealing a dynamic equilibrium between two functional states. Frequent scrunching/unscruching transitions and the imminent steric clashes of the inflating NT loop and growing RNA:DNA with the C-tail explain abortive synthesis and transition into elongation.
Asunto(s)
ADN Mitocondrial/genética , ARN Polimerasas Dirigidas por ADN/genética , Mitocondrias/genética , Proteínas Mitocondriales/genética , ARN Mitocondrial/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Sitios de Unión , Microscopía por Crioelectrón , ADN Mitocondrial/química , ADN Mitocondrial/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Motivos de Nucleótidos , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN Mitocondrial/química , ARN Mitocondrial/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Termodinámica , Elongación de la Transcripción Genética , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Iniciación de la Transcripción GenéticaRESUMEN
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).