Posse Viktor, Gustafsson Claes M
From the Department of Medical Biochemistry and Cell Biology, University of Gothenburg, P. O. Box 440, SE-405 30 Gothenburg, Sweden.
From the Department of Medical Biochemistry and Cell Biology, University of Gothenburg, P. O. Box 440, SE-405 30 Gothenburg, Sweden
J Biol Chem. 2017 Feb 17;292(7):2637-2645. doi: 10.1074/jbc.M116.751008. Epub 2016 Dec 27.
The mitochondrial transcription initiation machinery in humans consists of three proteins: the RNA polymerase (POLRMT) and two accessory factors, transcription factors A and B2 (TFAM and TFB2M, respectively). This machinery is required for the expression of mitochondrial DNA and the biogenesis of the oxidative phosphorylation system. Previous experiments suggested that TFB2M is required for promoter melting, but conclusive experimental proof for this effect has not been presented. Moreover, the role of TFB2M in promoter unwinding has not been discriminated from that of TFAM. Here we used potassium permanganate footprinting, DNase I footprinting, and transcription from the mitochondrial light-strand promoter to study the role of TFB2M in transcription initiation. We demonstrate that a complex composed of TFAM and POLRMT was readily formed at the promoter but alone was insufficient for promoter melting, which only occurred when TFB2M joined the complex. We also show that mismatch bubble templates could circumvent the requirement of TFB2M, but TFAM was still required for efficient initiation. Our findings support a model in which TFAM first recruits POLRMT to the promoter, followed by TFB2M binding and induction of promoter melting.
RNA聚合酶（POLRMT）和两个辅助因子，即转录因子A和B2（分别为TFAM和TFB2M）。该机制对于线粒体DNA的表达和氧化磷酸化系统的生物发生是必需的。先前的实验表明TFB2M是启动子解链所必需的，但尚未提供关于这种作用的确凿实验证据。此外，TFB2M在启动子解旋中的作用尚未与TFAM的作用区分开来。在这里，我们使用高锰酸钾足迹法、DNase I足迹法以及线粒体轻链启动子的转录来研究TFB2M在转录起始中的作用。我们证明由TFAM和POLRMT组成的复合物很容易在启动子处形成，但单独存在时不足以使启动子解链，只有当TFB2M加入该复合物时才会发生解链。我们还表明，错配气泡模板可以绕过对TFB2M的需求，但高效起始仍需要TFAM。我们的研究结果支持一种模型，即TFAM首先将POLRMT招募到启动子，随后TFB2M结合并诱导启动子解链。
