In Vitro-Reconstituted Nucleoids Can Block Mitochondrial DNA Replication and Transcription

• TFAM can compact DNA and reconstitute nucleoid-like structures in vitro
• TFAM packaging determines the number of active mtDNA molecules
• Nucleoid formation blocks mtDNA transcription and replication

SummaryThe mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000–10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, and at physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication.

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