Human mitochondrial transcription factor B1 interacts with the C-terminal activation region of h-mtTFA and stimulates transcription independently of its RNA methyltransferase activity
Human mitochondrial transcription factor B1 interacts with the C-terminal activation region of h-mtTFA and stimulates transcription independently of its RNA methyltransferase activity
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DOI：
10.1128/mcb.23.16.5816-5824.2003
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发表时间：
2003-08-01
影响因子：
5.3
通讯作者：
Shadel, GS
中科院分区：
文献类型：
--
作者：
McCulloch, V;Shadel, GS
在理解线粒体基因表达方面的一个重大进展是最近鉴定出两种新的人类线粒体转录因子h-mtTFB1和h-mtTFB2。这两种蛋白质都与高迁移率族盒转录因子h-mtTFA合作刺激转录，并且与rRNA甲基转移酶同源。事实上，最近通过其甲基化保守rRNA底物的能力证明了h-mtTFB1的双重功能性质。在这里，我们表明，h-mtTFB1结合h-mtTFA在HeLa细胞线粒体提取物和直接结合试验通过相互作用，需要的C-末端尾的h-mtTFA，转录激活所必需的区域。此外，点突变的保守甲基转移酶基序的h-mtTFB1显示，它刺激转录在体外独立的S-腺苷甲硫氨酸结合和rRNA甲基转移酶活性。此外，一个突变（G65A）消除了h-mtTFB1结合DNA的能力，但不影响转录激活。这些结果，再加上观察到的h-mtTFB1和人类线粒体RNA（h-mtDNA）聚合酶也可以coimmunoprecipitated，导致我们提出了一个模型，其中h-mtTFA划定线粒体启动子的位置和h-mtTFB蛋白桥接之间的相互作用的C-末端尾的h-mtTFA和mtDNA聚合酶，以促进特异性的转录起始。总而言之，这些数据提供了重要的新的见解，在人类线粒体的转录起始机制，并表明h-mtTFB1的双重功能可以分开。
A significant advancement in understanding mitochondrial gene expression is the recent identification of two new human mitochondrial transcription factors, h-mtTFB1 and h-mtTFB2. Both proteins stimulate transcription in collaboration with the high-mobility group box transcription factor, h-mtTFA, and are homologous to rRNA methyltransferases. In fact, the dual-function nature of h-mtTFB1 was recently demonstrated by its ability to methylate a conserved rRNA substrate. Here, we demonstrate that h-mtTFB1 binds h-mtTFA both in HeLa cell mitochondrial extracts and in direct-binding assays via an interaction that requires the C-terminal tail of h-mtTFA, a region necessary for transcriptional activation. In addition, point mutations in conserved methyltransferase motifs of h-mtTFB1 revealed that it stimulates transcription in vitro independently of S-adenosylmethionine binding and rRNA methyltransferase activity. Furthermore, one mutation (G65A) eliminated the ability of h-mtTFB1 to bind DNA yet did not affect transcriptional activation. These results, coupled with the observation that h-mtTFB1 and human mitochondrial RNA (h-mtRNA) polymerase can also be coimmunoprecipitated, lead us to propose a model in which h-mtTFA demarcates mitochondrial promoter locations and where h-mtTFB proteins bridge an interaction between the C-terminal tail of h-mtTFA and mtRNA polymerase to facilitate specific initiation of transcription. Altogether, these data provide important new insight into the mechanism of transcription initiation in human mitochondria and indicate that the dual functions of h-mtTFB1 can be separated.