SELECTION OF OPTIMAL KAPPA-B/REL DNA-BINDING MOTIFS - INTERACTION OF BOTH SUBUNITS OF NF-KAPPA-B WITH DNA IS REQUIRED FOR TRANSCRIPTIONAL ACTIVATION
SELECTION OF OPTIMAL KAPPA-B/REL DNA-BINDING MOTIFS - INTERACTION OF BOTH SUBUNITS OF NF-KAPPA-B WITH DNA IS REQUIRED FOR TRANSCRIPTIONAL ACTIVATION
复制标题
DOI：
10.1128/mcb.12.10.4412
复制
发表时间：
1992-10-01
影响因子：
5.3
通讯作者：
ROSEN, CA
中科院分区：
文献类型：
--
作者：
KUNSCH, C;RUBEN, SM;ROSEN, CA
对NF-κ B转录因子复合物的p50和p65亚基的分析表明，这两种蛋白质都可以通过同源二聚体或异源二聚体的形成与相关DNA序列相互作用。此外，原癌基因c-rel的产物可以通过自身或作为与p50或p65的异二聚体结合到类似的DNA基序。然而，这些研究使用了有限数量的已知kappaB DNA基序，并没有解决每个同源二聚体优选的最佳DNA序列的问题。使用纯化的重组p50、p65和c-Rel蛋白，从随机寡核苷酸池中选择最佳DNA结合基序。所选序列的比对使我们能够预测一个共识序列的个别同源二聚体的相关蛋白的结合，和DNA-蛋白结合分析所选的DNA序列揭示了序列特异性的蛋白质。与先前的假设相反，我们观察到p65同源二聚体可以与p50同源二聚体不识别的DNA序列的子集相互作用。还获得了与p50和c-Rel选择的序列的差异结合亲和力。使用p50-或p65-选择的kappaB基序，其显示相对于其他蛋白质的差异结合，几乎没有观察到与异源二聚体NF-kappaB复合物的结合。类似地，在使用选择性kappaB结合位点驱动氯霉素乙酰转移酶报告构建体表达的转染实验中，p65和p50选择性基序仅在p65和p50/65存在下被激活（具有p50 DNA结合结构域和p65活化结构域的嵌合蛋白）表达载体，也没有显示出对诱导NF-κ B活性的刺激的显著反应。这些发现表明，异源二聚体NF-κ B复合物的两个亚基与DNA的相互作用是DNA结合和转录激活所必需的，并表明由单个rel相关蛋白介导的转录激活将显着不同，这取决于存在的特定κ B基序。
Analysis of the p50 and p65 subunits of the NF-kappaB transcription factor complex has revealed that both proteins can interact with related DNA sequences through either homo- or heterodimer formation. In addition, the product of the proto-oncogene c-rel can bind to similar DNA motifs by itself or as a heterodimer with p50 or p65. However, these studies have used a limited number of known kappaB DNA motifs, and the question of the optimal DNA sequences preferred by each homodimer has not been addressed. Using purified recombinant p50, p65, and c-Rel proteins, optimal DNA-binding motifs were selected from a pool of random oligonucleotides. Alignment of the selected sequences allowed us to predict a consensus sequence for binding of the individual homodimeric Rel-related proteins, and DNA-protein binding analysis of the selected DNA sequences revealed sequence specificity of the proteins. Contrary to previous assumptions, we observed that p65 homodimers can interact with a subset of DNA sequences not recognized by p50 homodimers. Differential binding affinities were also obtained with p50- and c-Rel-selected sequences. Using either a p50- or p65-selected kappaB motif, which displayed differential binding with respect to the other protein, little to no binding was observed with the heterodimeric NF-kappaB complex. Similarly, in transfection experiments in which the selective kappaB binding sites were used to drive the expression of a chloramphenicol acetyltransferase reporter construct, the p65- and p50-selected motifs were activated only in the presence of p65 and p50/65 (a chimeric protein with the p50 DNA binding domain and p65 activation domain) expression vectors, respectively, and neither demonstrated a significant response to stimuli that induce NF-kappaB activity. These findings demonstrate that interaction of both subunits of the heterodimeric NF-kappaB complex with DNA is required for DNA binding and transcriptional activation and suggest that transcriptional activation mediated by the individual rel-related proteins will differ dramatically, depending on the specific kappaB motifs present.