Purification and enzymic properties of Mot1 ATPase, a regulator of basal transcription in the yeast Saccharomyces cerevisiae
Purification and enzymic properties of Mot1 ATPase, a regulator of basal transcription in the yeast Saccharomyces cerevisiae
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DOI：
10.1074/jbc.m002639200
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发表时间：
2000-07-14
影响因子：
4.8
通讯作者：
Thorner, J
中科院分区：
文献类型：
--
作者：
Adamkewicz, JI;Mueller, CGF;Thorner, J
Mot 1蛋白是ATP酶超家族的成员，其中一些是解旋酶，与蛋白质-核酸组装体相互作用。Mot 1是体内RNA聚合酶II依赖性转录的重要调节因子，并在体外解离TATA盒结合蛋白（TBP）-DNA复合物。Mot 1-（His）（6）从酵母提取物中纯化至表观均一。该制剂有效地解离TBP TATA复合物，表明不需要其他蛋白质或辅因子。Mot 1表现为一个非球状单体在流体动力学研究，差异标记的共表达Mot 1构建体之间没有检测到关联。ATP酶活性被高离子强度或碱性pH刺激约10倍，或通过删除N-末端TBP结合片段，表明N-末端结构域负调控C-末端ATP酶结构域（Mot 1C）。相应地，在中等盐浓度下，Mot 1 ATP酶（但不是Mot 1C）被酵母TBP刺激大于或等于10倍，这表明与TBP的相互作用解除了Mot 1中的构象约束。双链或单链TATA-含有DNA没有影响ATP酶活性的Mot 1或Mot 1C，有或没有TBP。Mot 1在使用具有平末端或5 '-或3'-突出端的底物的链置换测定中没有表现出可检测的解旋酶活性。Mot 1催化的TBP从DNA中的解离并没有被直接位于TATA序列之前的peptide交联所阻止。因此，Mot 1很可能通过引起TBP本身的结构变化而不是通过链解旋来促进TBP从含TATA的DNA中释放。
The 1867-residue Mot1 protein is a member of a superfamily of ATPases, some of which are helicases, that interact with protein-nucleic acid assemblies. Mot1 is an essential regulator of RNA polymerase II-dependent transcription in vivo and dissociates TATA box-binding protein (TBP)-DNA complexes in vitro. Mot1-(His)(6) was purified to apparent homogeneity from yeast extracts. The preparation efficiently dissociated TBP TATA complexes, suggesting that no other protein or cofactor is required. Mot1 behaved as a non-globular monomer in hydrodynamic studies, and no association was detected between differentially tagged co-expressed Mot1 constructs. ATPase activity was stimulated about 10-fold by high ionic strength or alkaline pH, or by deletion of the N-terminal TBP-binding segment, suggesting that the N-terminal domain negatively regulates the C-terminal ATPase domain (Mot1C). Correspondingly, at moderate salt concentration, Mot1 ATPase (but not Mot1C) was stimulated greater than or equal to 10-fold by yeast TBP, suggesting that interaction with TBP relieves a conformational constraint in Mot1. Double- or single-stranded TATA-containing DNA did not affect ATPase activity of Mot1 or Mot1C, with or without TBP. Mot1 did not exhibit detectable helicase activity in strand displacement assays using substrates with flush ends or 5'- or 3'-overhangs. Mot1-catalyzed dissociation of TBP from DNA was not prevented by a psoralen cross-link positioned immediately preceding the TATA sequence. Thus, Mot1 most likely promotes release of TBP from TATA-containing DNA by causing a structural change in TBP itself, rather than by strand unwinding.