Acetylation and phosphorylation of mitochondrial transcription factor a promote mitochondrial transcription processivity.

Abstract

Mitochondrial transcription factor A (TFAM) is the main component in mitochondrial nucleoids. TFAM serves as the mitochondrial DNA (mtDNA) compaction protein and as an essential transcription initiation factor. The compaction level of mtDNA has been proposed to regulate processes involving mtDNA like transcription and replication processivity. One mechanism to regulate TFAM binding to mtDNA within the mitochondria is via post-translational modifications (PTMs), such as phosphorylation by protein kinase A (PKA) and nonenzymatic acetylation by acetyl-CoA. Here, we demonstrate that DNA-bound TFAM is less susceptible to these modifications. We confirm using electrophoretic mobility shift assays that phosphorylated or acetylated TFAM compacted circular double-stranded DNA just as well as unmodified TFAM. Additionally, we provide an in-depth analysis of TFAM's lysine reactivity towards acetyl-CoA using isotopic labelling and liquid chromatography-tandem mass spectrometry. We show that both modifications to TFAM increase the processivity of transcription through TFAM-imposed barriers on DNA, but that TFAM bearing either modification maintains full activity in transcription initiation. We conclude that phosphorylation by PKA and nonenzymatic acetylation by acetyl-CoA are unlikely to occur on TFAM that are bound to the mitochondrial DNA. Instead, we propose that PTMs increase transcription output by modifying TFAM before it binds mtDNA. These findings call into question the PTM model of mitochondrial nucleoid regulation.