HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN-K IS A DNA-BINDING TRANSACTIVATOR
HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN-K IS A DNA-BINDING TRANSACTIVATOR
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DOI：
10.1074/jbc.270.9.4875
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发表时间：
1995-03-03
影响因子：
4.8
通讯作者：
LEVENS, D
中科院分区：
文献类型：
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作者：
TOMONAGA, T;LEVENS, D
我们以前曾报道，异质性核核糖核蛋白K（hnRNP K）结合到人类c-myc基因中发现的CT元件的富含嘧啶的链，并激活CT转录驱动的基因在体内的表达。我们现在描述了hnRNP K与CT元件相互作用所需的DNA和蛋白质。首先，hnRNP K被证明优先结合单链DNA而不是RNA或天然双链DNA。使用特异性寡核糖核苷酸或脱氧核糖核苷酸探针与特异性或非特异性RNA或DNA竞争物，电泳迁移率变动分析揭示hnRNP K：是DNA结合蛋白。特异性结合并不简单地反映结合到嘧啶丰富的序列的数量和排列的个别CT元素管理与hnRNP K的相互作用，至少有两个CT重复间隔至少三个核苷酸的结合是必需的，表明存在特定的立体化学约束调节CT-hnRNP K复合物的形成。缺失分析表明，hnRNP K具有几个不重叠的，DNA结合域，每个能够特异性：与CT元件结合，并优先DNA RNA。每个序列识别结构域由至少一个K同源基序组成，而hnRNP的较大部分可能是稳定的RNA结合所必需的。另外的实验表明，hnRNP K的N-末端35个残基是反式激活CT元件所必需的。这些结果表明hnRNP K是RNA结合蛋白和转录激活因子。
We have previously reported that heterogeneous nuclear ribonucleoprotein K (hnRNP K) binds to the pyrimidine-rich strand of the CT element found in the human c-myc gene and activates CT reporter-driven gene expression in vivo. We now characterize the DNA and protein requirements for the interaction of hnRNP K with the CT element, First, hnRNP K is shown to preferentially bind single-stranded DNA over RNA or native double-stranded DNA Using specific oligoribonucleotide or deoxyribonucleotide probes with specific or nonspecific RNA or DNA competitors, electrophoretic mobility shift assay revealed hnRNP K: to be a DNA-binding protein. Specific binding was not simply a reflection of binding to pyrimidine-rich sequences as the number and arrangement of individual CT elements governed interactions with hnRNP K; at least two CT repeats separated by at least three nucleotides are required for binding, indicating the existence of particular stereochemical constraints regulating CT-hnRNP K complex formation. Deletion analysis showed that hnRNP K possesses several nonoverlapping, DNA binding domains, each capable of specific: binding with the CT element and preferring DNA over RNA. Each sequence recognition domain is composed of at least one K homology motif, while a larger portion of hnRNP It may be required for stable RNA binding, Additional experiments indicate that the N-terminal 35 residues of hnRNP K are necessary for transactivating the CT element. These results indicate that hnRNP K is a RNA-binding protein and transcriptional activator.