PDF(Pubmed)
Abstract：
Mitochondrial transcription factor A (TFAM) employs DNA bending to package mitochondrial DNA (mtDNA) into nucleoids and recruit mitochondrial RNA polymerase (POLRMT) at specific promoter sites, light strand promoter (LSP) and heavy strand promoter (HSP). Herein, we characterize the conformational dynamics of TFAM on promoter and non-promoter sequences using single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule protein-induced fluorescence enhancement (smPIFE) methods. The DNA-TFAM complexes dynamically transition between partially and fully bent DNA conformational states. The bending/unbending transition rates and bending stability are DNA sequence-dependent-LSP forms the most stable fully bent complex and the non-specific sequence the least, which correlates with the lifetimes and affinities of TFAM with these DNA sequences. By quantifying the dynamic nature of the DNA-TFAM complexes, our study provides insights into how TFAM acts as a multifunctional protein through the DNA bending states to achieve sequence specificity and fidelity in mitochondrial transcription while performing mtDNA packaging.
摘要：
线粒体转录因子A（TFAM）利用DNA弯曲将线粒体DNA（mtDNA）包装成类核苷酸，并在特定的启动子位点招募线粒体RNA聚合酶（POLRMT），轻链启动子(LSP)和重链启动子(HSP)。在这里，我们使用单分子荧光共振能量转移（smFRET）和单分子蛋白质诱导的荧光增强（smPIFE）方法表征了TFAM在启动子和非启动子序列上的构象动力学。DNA-TFAM复合物在部分和完全弯曲的DNA构象状态之间动态地转变。弯曲/不弯曲过渡速率和弯曲稳定性是DNA序列依赖性的-LSP形成最稳定的完全弯曲复合物，而非特异性序列最少，这与TFAM与这些DNA序列的寿命和亲和力相关。通过定量DNA-TFAM复合物的动态性质，我们的研究提供了有关TFAM如何通过DNA弯曲状态充当多功能蛋白质的见解，以在线粒体转录中实现序列特异性和保真度，同时进行mtDNA包装。
