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首页> 外文期刊>Molecular and Cellular Biology >Functional characterization of the NF-kappa B p65 transcriptional activator and an alternatively spliced derivative.
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Functional characterization of the NF-kappa B p65 transcriptional activator and an alternatively spliced derivative.

机译：NF-κBp65转录激活剂和选择性剪接的衍生物的功能表征。

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The NF-kappa B transcription factor complex is composed of two proteins, designated p50 and p65, both having considerable homology to the product of the rel oncogene. We present evidence that the p65 subunit is a potent transcriptional activator in the apparent absence of the p50 subunit, consistent with in vitro results demonstrating that p65 can interact with DNA on its own. To identify the minimal activation domain, chimeric fusion proteins between the DNA binding domain of the yeast transcriptional activator protein GAL4 and regions of the carboxy terminus of p65 were constructed, and their transcriptional activity was assessed by using a GAL4 upstream activation sequence-driven promoter-chloramphenicol acetyltransferase fusion. This analysis suggests that the boundaries of the activation domain lie between amino acids 415 and 550. Moreover, single amino acid changes within residues 435 to 459 greatly diminished activation. Similar to other activation domains, this region contains a leucine zipper-like motif as well as an overall net negative charge. To identify those residues essential for DNA binding, we made use of a naturally occurring derivative of p65, lacking residues 222 to 231 (hereafter referred to as p65 delta), and produced via an alternative splice site. Gel mobility shift analysis using bacterially expressed p65, p65 delta, and various mutants indicates that residues 222 to 231 are important for binding to kappa B DNA. Coimmunoprecipitation analysis suggests that these residues likely contribute to the multimerization function required for homomeric complex formation or heteromeric complex formation with p50 in that no association of p65 delta with itself or with p50 was evident. However, p65 delta was able to form weak heteromeric complexes with p65 that were greatly reduced in their ability to bind DNA. On the basis of these findings, we suggest that subtle changes within the proposed multimerization domain can elicit different effects with the individual Rel-related proteins and that a potential role of p65 delta may be to negatively regulate NF-kappa B function through formation of nonfunctional heteromeric complexes.
机译：NF-κB转录因子复合物由两种蛋白质组成，分别命名为p50和p65，两者均与rel癌基因的产物具有相当的同源性。我们提供的证据表明，在明显缺乏p50亚基的情况下，p65亚基是一种有效的转录激活因子，这与证明p65可以自身与DNA相互作用的体外结果一致。为了确定最小的激活结构域，构建了酵母转录激活蛋白GAL4的DNA结合结构域与p65羧基末端区域之间的嵌合融合蛋白，并使用GAL4上游激活序列驱动的启动子-氯霉素乙酰转移酶融合。该分析表明活化结构域的边界位于氨基酸415和550之间。此外，残基435至459内的单个氨基酸变化大大降低了活化。与其他激活域相似，该区域包含亮氨酸拉链样基序以及总的净负电荷。为了鉴定对于DNA结合必不可少的那些残基，我们利用了天然存在的p65衍生物，该残基缺少残基222至231（以下称为p65δ），并通过替代的剪接位点产生。使用细菌表达的p65，p65δ和各种突变体进行的凝胶迁移率迁移分析表明，残基222至231对于与κB DNA结合很重要。免疫共沉淀分析表明，这些残基可能有助于形成与p50形成同聚复合物或形成异聚复合物所需的多聚功能，因为没有明显的p65δ与自身或与p50的缔合。但是，p65δ能够与p65形成弱的异聚复合物，从而大大降低了其结合DNA的能力。根据这些发现，我们建议拟议的多聚化结构域内的细微变化可引起与单个Rel相关蛋白的不同作用，并且p65δ的潜在作用可能是通过形成非功能性负调控NF-κB功能。异聚体。

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