Department of Physics and Astronomy and LaserLaB, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands.
Department of Microbiology, Biochemistry and Molecular Genetics, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 07103, USA.
Nucleic Acids Res. 2018 Apr 20;46(7):3633-3642. doi: 10.1093/nar/gky204.
Mitochondrial transcription factor A (TFAM) is essential for the maintenance, expression and transmission of mitochondrial DNA (mtDNA). However, mechanisms for the post-translational regulation of TFAM are poorly understood. Here, we show that TFAM is lysine acetylated within its high-mobility-group box 1, a domain that can also be serine phosphorylated. Using bulk and single-molecule methods, we demonstrate that site-specific phosphoserine and acetyl-lysine mimics of human TFAM regulate its interaction with non-specific DNA through distinct kinetic pathways. We show that higher protein concentrations of both TFAM mimics are required to compact DNA to a similar extent as the wild-type. Compaction is thought to be crucial for regulating mtDNA segregation and expression. Moreover, we reveal that the reduced DNA binding affinity of the acetyl-lysine mimic arises from a lower on-rate, whereas the phosphoserine mimic displays both a decreased on-rate and an increased off-rate. Strikingly, the increased off-rate of the phosphoserine mimic is coupled to a significantly faster diffusion of TFAM on DNA. These findings indicate that acetylation and phosphorylation of TFAM can fine-tune TFAM-DNA binding affinity, to permit the discrete regulation of mtDNA dynamics. Furthermore, our results suggest that phosphorylation could additionally regulate transcription by altering the ability of TFAM to locate promoter sites.
线粒体转录因子 A（TFAM）对于线粒体 DNA（mtDNA）的维持、表达和传递是必不可少的。然而，TFAM 的翻译后调控机制还知之甚少。在这里，我们发现 TFAM 在其高迁移率族蛋白 1 内发生赖氨酸乙酰化，该结构域也可以丝氨酸磷酸化。使用批量和单分子方法，我们证明了人 TFAM 的特异性磷酸丝氨酸和乙酰化赖氨酸模拟物通过不同的动力学途径调节其与非特异性 DNA 的相互作用。我们表明，两种 TFAM 模拟物的更高蛋白浓度都需要将 DNA 压缩到与野生型相似的程度。压缩被认为对于调节 mtDNA 的分离和表达至关重要。此外，我们揭示了乙酰化赖氨酸模拟物的 DNA 结合亲和力降低是由于进入速率较低，而磷酸丝氨酸模拟物则显示出进入速率降低和离开速率增加。引人注目的是，磷酸丝氨酸模拟物的增加离开速率与 TFAM 在 DNA 上的更快扩散相关。这些发现表明，TFAM 的乙酰化和磷酸化可以微调 TFAM-DNA 结合亲和力，从而允许对 mtDNA 动力学进行离散调节。此外，我们的结果表明，磷酸化还可以通过改变 TFAM 定位启动子位点的能力来调节转录。
