PURIFICATION AND CHARACTERIZATION OF HUMAN MITOCHONDRIAL TRANSCRIPTION FACTOR-I
PURIFICATION AND CHARACTERIZATION OF HUMAN MITOCHONDRIAL TRANSCRIPTION FACTOR-I
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DOI：
10.1128/mcb.8.8.3496
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发表时间：
1988-08-01
影响因子：
5.3
通讯作者：
CLAYTON, DA
中科院分区：
文献类型：
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作者：
FISHER, RP;CLAYTON, DA
我们从人KB细胞线粒体中提纯了一种近乎同源的转录因子。该因子被命名为线粒体转录1(MtTF1)，是同源线粒体RNA聚合酶体外识别人类线粒体DNA的两个主要启动子所必需的。此外，它已被证明结合了两个主要启动子的上游调控成分。用磷酸纤维素层析法从RNA聚合酶中分离mtTF1后，在MonoQ阴离子交换快速高效液相色谱柱上对mtTF1进行层析。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法对mtTF1-组分进行分析，发现了单一的主要多肽，其MR约为25000。甘油分析梯度离心法测得沉淀系数为.apprx。S，与25kodalton的单体蛋白一致。最后，当从染色的十二烷基硫酸钠-聚丙烯酰胺凝胶中取出25kodalton的多肽并允许复性时，它在两个启动子上都重新获得了DNA结合和转录刺激活性。虽然mtTF1是目前唯一纯化和鉴定的线粒体DNA结合转录因子，但其对随机DNA的高亲和力和对其靶序列的弱特异性可能是这类调控蛋白的典型特征。
We purified to near homogeneity a transcription factor from human KB cell mitochondria. This factor, designated mitochondrial transcription 1 (mtTF1), is required for the in vitro recognition of both major promoters of human mitochondrial DNA by the homologous mitochondrial RNA polymerase. Furthermore, it has been shown to bind upstream regulatory elements of the two major promoters. After separation from RNA polymerase by phosphocellulose chromatography, mtTF1 was chromatographed on a MonoQ anion-exchange fast-performance liquid chromatography column. Analysis of mtTF1-containing fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single major polypeptide with an Mr of approximately 25,000. Centrifugation in analytical glycerol gradients indicated a sedimentation coefficient of .apprx. 2.5 S, consistent with a monomeric 25-kilodalton protein. Finally, when the 25-kilodalton polypeptide was excised from a stained sodium dodecyl sulfate-polyacrylamide gel and allowed to renature, it regained DNA-binding and transcriptional stimulatory activities at both promoters. Although mtTF1 is the only mitochondrial DNA-binding transcription factor to be purified and characterized, its properties, such as a high affinity for random DNA and a weak specificity for one of its target sequences, may typify this class of regulatory proteins.