The structurally homologous Mtf1 and TFB2M proteins serve as transcription initiation factors of mitochondrial RNA polymerases in Saccharomyces cerevisiae and humans, respectively. These transcription factors directly interact with the nontemplate strand of the transcription bubble to drive promoter melting. Given the key roles of Mtf1 and TFB2M in promoter-specific transcription initiation, it can be expected that the DNA binding activity of the mitochondrial transcription factors is regulated to prevent DNA binding at inappropriate times. However, little information is available on how mitochondrial DNA transcription is regulated. While studying C-terminal (C-tail) deletion mutants of Mtf1 and TFB2M, we stumbled upon a finding that suggested that the flexible C-tail region of these factors autoregulates their DNA binding activity. Quantitative DNA binding studies with fluorescence anisotropy-based titrations revealed that Mtf1 with an intact C-tail has no affinity for DNA but deletion of the C-tail greatly increases Mtf1's DNA binding affinity. Similar observations were made with TFB2M, although autoinhibition by the C-tail of TFB2M was not as complete as in Mtf1. Analysis of available TFB2M structures disclosed that the C-tail engages in intramolecular interactions with the DNA binding groove in the free factor, which, we propose, inhibits its DNA binding activity. Further experiments showed that RNA polymerase relieves this autoinhibition by interacting with the C-tail and engaging it in complex formation. In conclusion, our biochemical and structural analyses reveal autoinhibitory and activation mechanisms of mitochondrial transcription factors that regulate their DNA binding activities and aid in specific assembly of transcription initiation complexes.

中文翻译：

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线粒体转录因子Mtf1和TFB2M的C末端尾巴是调节DNA结合的自抑制机制的一部分。

结构同源的Mtf1和TFB2M蛋白分别作为酿酒酵母和人中线粒体RNA聚合酶的转录起始因子。这些转录因子直接与转录气泡的非模板链相互作用，以驱动启动子融化。鉴于Mtf1和TFB2M在启动子特异性转录起始中的关键作用，可以预期调节线粒体转录因子的DNA结合活性以防止在不适当的时间结合DNA。但是，关于线粒体DNA转录调控的信息很少。在研究Mtf1和TFB2M的C末端（C-tail）缺失突变体时，我们偶然发现了一个发现，表明这些因子的柔性C-tail区域会自动调节其DNA结合活性。基于荧光各向异性滴定的定量DNA结合研究表明，具有完整C-tail的Mtf1对DNA没有亲和力，但C-tail的缺失大大增加了Mtf1的DNA结合亲和力。对于TFB2M也有类似的观察结果，尽管TFB2M的C尾自动抑制作用不如Mtf1中的完全。对可用TFB2M结构的分析表明，C尾与游离因子中的DNA结合槽参与分子内相互作用，我们提议抑制其DNA结合活性。进一步的实验表明，RNA聚合酶通过与C尾相互作用并使之参与复合物形成，从而缓解了这种自抑制作用。结论，