ANALYSIS OF ARGININE-RICH PEPTIDES FROM THE HIV TAT PROTEIN REVEALS UNUSUAL FEATURES OF RNA PROTEIN RECOGNITION
ANALYSIS OF ARGININE-RICH PEPTIDES FROM THE HIV TAT PROTEIN REVEALS UNUSUAL FEATURES OF RNA PROTEIN RECOGNITION
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DOI：
10.1101/gad.5.2.201
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发表时间：
1991-02-01
影响因子：
10.5
通讯作者：
FRANKEL, AD
中科院分区：
文献类型：
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作者：
CALNAN, BJ;BIANCALANA, S;FRANKEL, AD
富含精氨酸的序列存在于许多RNA结合蛋白中，并被认为可介导特异性RNA识别。HIV - 1 Tat蛋白含有Tat富含精氨酸区域的片段能特异性结合TAR RNA中的一个3核苷酸凸起。为确定特异性RNA识别对氨基酸的要求，我们合成了一系列跨越该结构域（YGRKKRRQRRRP）的突变Tat肽，并测量了它们对TAR RNA的亲和力和特异性。将几个相应的突变引入全长Tat蛋白，并测量其反式激活活性。将精氨酸残基系统地替换为丙氨酸或赖氨酸表明，总体电荷密度很重要，但未指出任何对结合至关重要的特定残基。谷氨酰胺替换为丙氨酸对结合没有影响。值得注意的是，序列打乱或颠倒的肽对TAR RNA表现出与野生型肽相同的亲和力和特异性。突变Tat蛋白的反式激活活性与RNA结合相关。来自SIV Tat以及HIV - 1 Rev的富含精氨酸的肽（它们可在功能上替代HIV - 1 Tat的碱性区域）也能特异性结合TAR。圆二色性光谱表明，在没有RNA时，Tat的富含精氨酸区域无结构，结合时会部分或完全形成结构，并诱导RNA发生构象变化。这些结果表明，富含精氨酸的RNA结合结构域具有相当大的序列灵活性，这让人联想到转录激活因子中的酸性结构域，并且RNA结构可能为这种相互作用提供了大部分特异性。
Arginine-rich sequences are found in many RNA-binding proteins and have been proposed to mediate specific RNA recognition. Fragments of the HIV-1 Tat protein that contain the arginine-rich region of Tat bind specifically to a 3-nucleotide bulge in TAR RNA. To determine the amino acid requirements for specific RNA recognition, we synthesized a series of mutant Tat peptides spanning this domain (YGRKKRRQRRRP) and measured their affinity and specificity for TAR RNA. Several corresponding mutations were introduced into the full-length Tat protein, and trans-activation activity was measured. Systematic substitution of arginine residues with alanines or lysines suggested that overall charge density is important but did not point to any specific residues as being essential for binding. A glutamine-to-alanine substitution had no effect on binding. Remarkably, peptides with scrambled or reversed sequences showed the same affinity and specificity for TAR RNA as the wild-type peptide. Trans-activation activity of the mutant Tat proteins correlated with RNA binding. Arginine-rich peptides from SIV Tat and from HIV-1, Rev, which can functionally substitute for the basic region of HIV-1 Tat, also bound specifically to TAR. Circular dichroism spectra suggest that the arginine-rich region of Tat is unstructured in the absence of RNA, becomes partially or fully structured upon binding, and induces a conformational change in the RNA. These results suggest that arginine-rich RNA-binding domains have considerable sequence flexibility, reminiscent of acidic domains found in transcriptional activators, and that RNA structure may provide much of the specificity for the interaction.