Human mitochondrial transcription factors TFAM and TFB2M work synergistically in promoter melting during transcription initiation.
Human mitochondrial transcription factors TFAM and TFB2M work synergistically in promoter melting during transcription initiation.
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DOI：
10.1093/nar/gkw1157
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发表时间：
2017-01-25
影响因子：
14.9
通讯作者：
Patel SS
中科院分区：
文献类型：
--
作者：
Ramachandran A;Basu U;Sultana S;Nandakumar D;Patel SS
人线粒体DNA由POLRMT在两个起始因子TFAM和TFB 2 M的帮助下转录。目前的模型假定TFAM的作用是募集POLRMT和TFB 2 M以熔化启动子。然而，我们发现，TFAM有“后招聘”的作用，在启动子熔化和RNA合成，这是通过研究启动子结合，弯曲和熔化，和流产的RNA合成的前起始步骤。我们的2-氨基嘌呤图谱研究表明，LSP（轻链启动子）在与所有三种蛋白质的开放复合物中从-4解链到+1，在加入ATP后从-4解链到+3。我们的平衡结合研究表明，POLRMT形成稳定的复合物与TFB 2 M或TFAM LSP具有低纳摩尔Kd值，但这些双组分复合物缺乏有效地熔化的启动子的机制。这表明POLRMT需要TFB 2 M和TFAM两者来熔化启动子。此外，POLRMT+ TFB 2 M在LSP上产生2-mer的败育体，但仅用TFAM观察到更长的RNA。这些结果被解释TFAM在启动子熔化和/或LSP上的开放复合物的稳定中起作用。基于我们的研究结果，我们提出了一个完善的模型，转录起始的人线粒体转录机制。
Human mitochondrial DNA is transcribed by POLRMT with the help of two initiation factors, TFAM and TFB2M. The current model postulates that the role of TFAM is to recruit POLRMT and TFB2M to melt the promoter. However, we show that TFAM has ‘post-recruitment’ roles in promoter melting and RNA synthesis, which were revealed by studying the pre-initiation steps of promoter binding, bending and melting, and abortive RNA synthesis. Our 2-aminopurine mapping studies show that the LSP (Light Strand Promoter) is melted from −4 to +1 in the open complex with all three proteins and from −4 to +3 with addition of ATP. Our equilibrium binding studies show that POLRMT forms stable complexes with TFB2M or TFAM on LSP with low-nanomolar Kd values, but these two-component complexes lack the mechanism to efficiently melt the promoter. This indicates that POLRMT needs both TFB2M and TFAM to melt the promoter. Additionally, POLRMT+TFB2M makes 2-mer abortives on LSP, but longer RNAs are observed only with TFAM. These results are explained by TFAM playing a role in promoter melting and/or stabilization of the open complex on LSP. Based on our results, we propose a refined model of transcription initiation by the human mitochondrial transcription machinery.