Oxytricha telomere-binding protein: separable DNA-binding and dimerization domains of the alpha-subunit.
Oxytricha telomere-binding protein: separable DNA-binding and dimerization domains of the alpha-subunit.
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尖毛虫端粒结合蛋白：α 亚基的可分离 DNA 结合和二聚化结构域。
DOI：
10.1101/gad.7.5.870
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发表时间：
1993
影响因子：
10.5
通讯作者：
Cech,TR
中科院分区：
文献类型：
--
作者：
Fang,G;Gray,JT;Cech,TR
56-kD (α) 和 41-kD (β) 亚基的端粒结合蛋白异二聚体与每个 Oxytricha nova 大核 DNA 分子的单链 (T4G4)2 末端结合。 α 亚基本身与端粒 DNA 结合。单独的β亚基不与DNA特异性结合，但与α亚基相互作用形成非常稳定的三元复合物。我们表明，α-β-DNA 三元复合物的形成是极其合作的。此外，二元复合物（α-DNA）的解离半衰期远小于 1 分钟；添加 β 亚基可将半衰期延长至约 100 小时。将随机删除 α 亚基开放阅读框的质粒文库引入大肠杆菌，随后检查提取物的蛋白质表达和 DNA 结合活性（添加或不添加 β 亚基）。发现α亚基包含两个结构上可分离的具有不同功能的结构域。氨基末端的三分之二对于序列特异性 DNA 结合是必要且充分的。羧基末端的三分之一负责α/β亚基相互作用。当在大肠杆菌中单独表达、纯化并混合在一起时，这两个结构域重建了野生型α亚基的活性（体外反式互补）。 β 亚基的氨基末端三分之二对于 α/β 亚基相互作用和三元复合物形成都是必要且充分的。我们得出的结论是，端粒结合蛋白的 α 亚基与许多转录因子一样，具有可分离的 DNA 结合域和蛋白质-蛋白质相互作用域。
A telomere-binding protein heterodimer of 56-kD (alpha) and 41-kD (beta) subunits binds to the single-stranded (T4G4)2 terminus of each Oxytricha nova macronuclear DNA molecule. The alpha-subunit by itself binds to telomeric DNA. The beta-subunit alone does not bind to DNA specifically but interacts with the alpha-subunit to form a very stable ternary complex. We show that the formation of alpha-beta-DNA ternary complex is extremely cooperative. Furthermore, the binary complex (alpha-DNA) has a dissociation half-life of much less than 1 min; addition of the beta-subunit increases the half-life to approximately 100 hrs. Libraries of plasmids with random deletions of the open reading frame for the alpha-subunit were introduced into Escherichia coli, and extracts were subsequently checked for both protein expression and DNA-binding activity with or without added beta-subunit. The alpha-subunit was found to contain two structurally separable domains with distinct functions. The amino-terminal two-thirds is necessary and sufficient for sequence-specific DNA binding. The carboxy-terminal one-third is responsible for alpha/beta-subunit interactions. When expressed separately in E. coli, purified, and mixed together, these two domains reconstitute the activity of the wild-type alpha-subunit (trans-complementation in vitro). The amino-terminal two-thirds of the beta-subunit is necessary and sufficient both for alpha/beta-subunit interactions and for ternary complex formation. We conclude that the alpha-subunit of the telomere-binding protein, like many transcription factors, has separable DNA-binding and protein-protein interaction domains.