Characterisation of the nucleic-acid-binding activity of KH domains - Different properties of different domains
Characterisation of the nucleic-acid-binding activity of KH domains - Different properties of different domains
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DOI：
10.1111/j.1432-1033.1996.00425.x
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发表时间：
1996-10-15
期刊：
影响因子：
--
通讯作者：
Leffers, H
中科院分区：
文献类型：
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作者：
Dejgaard, K;Leffers, H
KH 模块是最近在多种 RNA 结合蛋白中发现的序列基序，并被认为是负责 RNA 结合的功能元件。然而到目前为止，这一假设还没有得到直接的实验支持。我们表达了异质核核糖核蛋白 K (hnRNP-K) 的三个 KH 结构域、聚 (C) 结合蛋白 PCBP-1 和 PCBP-2、高密度结合蛋白 HBP 的前三到四个结构域、古细菌盐杆菌 ORF139 的一个半结构域以及脆弱 X 蛋白的一个半结构域 大肠杆菌中的 FMR1 并分析了它们的体外核酸结合特性。结果表明，hnRNP-K 的体外聚 (rC) 结合活性可归因于 KH 结构域 3，而 PCBP 中的结构域 1 和 3 均结合聚 (rC)。此外，所有这些结构域都表现出对其他核酸的结合活性，尽管其水平显着较低。 FMR1 蛋白的第一个 KH 结构域结合聚 (rG) 以及单链和双链 DNA。 HBP 的 N 端三个或四个结构域结合聚 (rG)，并以低得多的水平结合单链和双链 DNA。因此，单个KH结构域是离散且独立的核酸结合单元。而且。不同的 KH 结构域结合不同的核酸，这表明 KH 结构域由保守的、弱核酸结合的结构组成，该结构通过序列变异进行微调，从而产生序列特异性的核酸结合实体。
The KH module is a sequence motif recently identified in a number of diversified RNA-binding proteins and suggested to be the functional element responsible for RNA binding. So far, however, this hypothesis has not received direct experimental support. We have expressed the three KH-domains from heterogeneous nuclear ribonucleoprotein K (hnRNP-K), the poly(C)-binding proteins PCBP-1 and PCBP-2, the first three to four domains from the high-density binding protein HBP, the one and a half domain from the archaeon Halobacterium halobium ORF139 and one and a half domain of the fragile-X protein FMR1 in Escherichia coli and analysed their nucleic-acid-binding properties in vitro. The results showed that the in vitro poly(rC)-binding activity of hnRNP-K can be assigned to KH-domain 3, whereas both domains 1 and 3 in the PCBPs bind poly(rC). In addition, all these domains exhibit binding activity towards other nucleic acids, albeit at a significantly lower level. The first KH domain from the FMR1 protein binds poly(rG) and single-stranded and double-stranded DNA. The N-terminal three or four domains from HBP bind poly(rG) and, at a much lower level, single-stranded and double-stranded DNA. Thus, single KH domains are discrete and independent nucleic-acid-binding units. Moreover. different KH domains bind different nucleic acids, suggesting that KH domains are composed of a conserved, weakly nucleic-acid-binding, structure that is fine tuned, by sequence variation, resulting in sequence-specific nucleic-acid-binding entities.