The Role of Phosphorylation and Acetylation of TFAM in DNA Binding Regulation using Single-Molecule Manipulation and Fluorescence Microscopy

Abstract

Mitochondrial transcription factor A (TFAM) is a multifunctional protein, which orchestrates mitochondrial DNA compaction, transcription and replication. While post-translational modifications, such as TFAM phosphorylation and acetylation are thought to regulate these processes, the mechanism by which this regulation occurs is poorly understood.Using a combination of single-molecule manipulation and fluorescence microscopy, we investigate the effect of TFAM phosphorylation and acetylation on DNA binding affinity. By fitting force-extension curves of TFAM-bound DNA to the Worm-Like Chain model, we determine how the persistence length of DNA changes with increasing protein concentration. In this way, we determine the binding affinity of TFAM to DNA as well as the extent of TFAM induced DNA compaction. We demonstrate that phosphorylation and acetylation of TFAM do not alter its ability to compact DNA, but significantly lower the binding affinity to DNA.Furthermore, by visualizing fluorescently-labelled TFAM unbinding from DNA, we reveal an increase in the unbinding rate of TFAM from DNA upon phosphorylation. This indicates that the reduced binding affinity of TFAM to DNA when phosphorylated is at least partially due to the higher off-rate of phosphorylated TFAM. Conversely, the unbinding rate of TFAM from DNA remains unaffected by acetylation. Therefore, we relate the lower binding affinity of acetylated TFAM to a decrease in the on-rate of the protein.These findings indicate that phosphorylation and acetylation can regulate TFAM function and may lead to a deeper understanding of the in vivo variations of TFAM coating on DNA and its exact biological function.