Assembly of b/HLH/z proteins c-Myc, Max, and Mad1 with cognate DNA: Importance of protein-protein and protein-DNA interactions
Assembly of b/HLH/z proteins c-Myc, Max, and Mad1 with cognate DNA: Importance of protein-protein and protein-DNA interactions
复制标题
DOI：
10.1021/bi050206i
复制
发表时间：
2005-09-06
期刊：
影响因子：
2.9
通讯作者：
Goss, DJ
中科院分区：
文献类型：
--
作者：
Hu, JZ;Banerjee, A;Goss, DJ
在转录因子中最好表征的是B/HLH/z蛋白：USF、Max、Mye和Mad。这些蛋白质结合到DNA E-box，一个六碱基对序列，CACGTG。Max和Myc形成具有强致癌潜力但也可以抑制转录的异源二聚体，而Mad和Max形成充当转录阻遏物的异源二聚体。我们已经使用荧光各向异性来测量蛋白质-蛋白质和蛋白质-DNA亲和力。MLP DNA与Max之间的特异性结合（K = 2.2 +/- 0.5 nM）的亲和力比LCR DNA高约10倍，比非特异性DNA高约100倍。USF与Max对MLP DNA的结合亲和力相似（K = 15 +/- 10 nM），但Max与LCR和非特异性DNA的结合更紧密。构建了一系列命名为E-box、半E-box和非E-box的寡核苷酸，以检查DNA序列的影响。结合结果表明，对于Max蛋白，大部分结合能可以归因于单个元素，在E-box的两个半部分之间具有很小的协同性。进一步的研究测量了单体-二聚体-DNA相互作用的整个热力学循环的平衡。令人惊讶的是，Max单体-DNA对第二单体的亲和力大大降低（第一单体的K在纳摩尔范围内，第二单体的K在微摩尔范围内）。从Max蛋白的角度来看，DNA与Max的结合显著降低了Max蛋白对第二单体的亲和力，无论第二单体是Myc、Mad还是Max。这些数据表明蛋白质-蛋白质相互作用在转录起始复合物组装中的重要性。
Among the best characterized of the transcription factors are the b/HLH/z proteins: USF, Max, Mye, and Mad. These proteins bind to the DNA E-box, a six base pair sequence, CACGTG. Max and Myc form a heterodimer that has strong oncogenic potential but can also repress transcription, while Mad and Max form a heterodimer that acts as a transcription repressor. We have used fluorescence anisotropy to measure protein-protein and protein-DNA affinity. The specific binding between MLP DNA and Max (K = 2.2 +/- 0.5 nM) is about 10-fold higher affinity than LCR DNA and about 100-fold higher than for a nonspecific DNA. USF has a similar binding affinity as Max to MLP DNA (K = 15 +/- 10 nM), but Max binds more tightly to LCR and nonspecific DNA. A series of oligonucleotides designated E-box, half-E-box, and non-E-box were constructed to examine the effects of DNA sequence. The binding results indicate that for Max protein most of the binding energy can be attributed to individual elements with little cooperativity among the two halves of the E-box. Further studies measured the equilibria for the entire thermodynamic cycle of monomer-dimer-DNA interactions. Surprisingly, the affinity of the Max monomer-DNA for the second monomer was greatly reduced (K for the first monomer in the nanomolar range and for the second monomer in the micromolar range). Looked at from the perspective of the Max protein, the binding of DNA to Max significantly reduces the affinity of the Max protein for the second monomer, whether the second monomer is Myc, Mad, or Max. These data suggest the importance of protein-protein interactions in assembly of a transcription initiation complex.