Mutations in TFIIIA that increase stability of the TFIIIA-5 S rRNA gene complex - Unusual effects on the kinetics of complex assembly and dissociation
Mutations in TFIIIA that increase stability of the TFIIIA-5 S rRNA gene complex - Unusual effects on the kinetics of complex assembly and dissociation
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DOI：
10.1074/jbc.m502677200
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发表时间：
2005-07-22
影响因子：
4.8
通讯作者：
Setzer, DR
中科院分区：
文献类型：
--
作者：
Brady, KL;Ponnampalam, SN;Setzer, DR
我们在非洲爪蟾 TFIIIA 中发现了四种突变，这些突变增加了 TFIIIA-5 S rRNA 基因复合物的稳定性。在每种情况下，突变对平衡结合亲和力的影响相对较小。在三种情况下，这些平衡结合效应主要归因于蛋白质-DNA 复合物解离速率常数的降低。然而，在第四种情况中，用苯丙氨酸取代 TFIIIA 中第 148 位的野生型亮氨酸会导致复合物组装和解离动力学发生更大的补偿性变化。这些数据支持这样一种模型，即具有多组分解离动力学的相对不稳定的复合物群体迅速形成；然后复合物经历缓慢的构象变化，形成非常稳定、动力学均质的 TFIIIA-DNA 复合物。当 L148F 突变蛋白与 VP16 激活结构域融合并在酵母细胞中表达时，它可作为一种特别有效的转录激活剂。将 L148 替换为酪氨酸或色氨酸会产生同样强的转录激活剂。组氨酸取代所产生的遗传和生化效应比 L148F 突变观察到的效应更温和，但与 L148F 突变相似。我们提出，在位置 148 处具有平面侧链的氨基酸可以插入 5S rRNA 基因中间元件中的相邻碱基对之间。嵌入发生缓慢，但会产生非常稳定的 DNA-蛋白质复合物。这些结果表明，顺式作用序列元件的转录激活很大程度上取决于与激活蛋白形成的复合物的动力学稳定性，而不是热力学稳定性。因此，转录激活在很大程度上取决于激活剂-DNA复合物的寿命，而不是稳定状态下结合位点的占用。将嵌入氨基酸引入锌指蛋白可能是生产具有特别高的体内活性的人工转录因子的有用工具。
We have identified four mutations in Xenopus TFIIIA that increase the stability of TFIIIA-5 S rRNA gene complexes. In each case, the mutation has a relatively modest effect on equilibrium binding affinity. In three cases, these equilibrium binding effects can be ascribed primarily to decreases in the rate constant for protein-DNA complex dissociation. In the fourth case, however, a substitution of phenylalanine for the wild-type leucine at position 148 in TFIIIA results in much larger compensating changes in the kinetics of complex assembly and dissociation. The data support a model in which a relatively unstable population of complexes with multi-component dissociation kinetics forms rapidly; complexes then undergo a slow conformational change that results in very stable, kinetically homogeneous TFIIIA-DNA complexes. The L148F mutant protein acts as a particularly potent transcriptional activator when it is fused to the VP16 activation domain and expressed in yeast cells. Substitution of L148 to tyrosine or tryptophan produces an equally strong transcriptional activator. Substitution to histidine results in genetic and biochemical effects that are more modest than, but similar to, those observed with the L148F mutation. We propose that an amino acid with a planar side chain at position 148 can intercalate between adjacent base pairs in the intermediate element of the 5 S rRNA gene. Intercalation occurs slowly but results in a very stable DNA-protein complex. These results suggest that transcriptional activation by a cis-acting sequence element is largely dependent on the kinetic, rather than the thermodynamic, stability of the complex formed with an activator protein. Thus, transcriptional activation is dependent in large part on the lifetime of the activator-DNA complex rather than on binding site occupancy at steady state. Introduction of intercalating amino acids into zinc finger proteins may be a useful tool for producing artificial transcription factors with particularly high in vivo activity.