Abstract
Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Conformational transitions of the transcription initiation complex must be central for such control, but how the conformational dynamics progress throughout transcription initiation remains unknown. Here, we use single-molecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the transcriptional system of yeast mitochondria with single-base resolution. We show that the yeast mitochondrial transcriptional complex dynamically transitions among closed, open, and scrunched states throughout the initiation stage. Then abruptly at position +8, the dynamic states of initiation make a sharp irreversible transition to an unbent conformation with associated promoter release. Remarkably, stalled initiation complexes remain in dynamic scrunching and unscrunching states without dissociating the RNA transcript, implying the existence of backtracking transitions with possible regulatory roles. The dynamic landscape of transcription initiation suggests a kinetically driven regulation of mitochondrial transcription.
Highlights
Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism
Our results reveal conformational dynamics during mitochondrial transcription initiation that could be vital checkpoints for regulating mitochondrial transcription efficiency and promoter selection
In vitro transcription assays confirmed that these template designs produce the expected abortive transcripts at each stalling position[43]
Summary
Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Transcriptional systems across lineages show highly variable efficiencies of RNA initiation that depend on specific DNA elements[12,13] Both the initiation stage and transition to elongation are regulatory targets of many proteins and small molecules[10,14]. Mtf[1] is structurally and functionally homologous to TFB2M21,24, and functions like the bacterial sigma factors[25,26,27] These transcription systems have developed a set of molecular mechanisms to regulate transcription efficiency. Both bacteriophage and bacterial RNAPs show scrunching of the downstream DNA into the active site as the RNA–DNA hybrid, and transcription bubble grows during initiation. Branching and pausing between competing pathways has been observed in bacteriophages and bacteria that may represent targets of regulation for the transcription activity[3,7,8]
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