ROLE OF RNA STRUCTURE IN ARGININE RECOGNITION OF TAR RNA
ROLE OF RNA STRUCTURE IN ARGININE RECOGNITION OF TAR RNA
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DOI：
10.1073/pnas.90.8.3680
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发表时间：
1993-04-15
影响因子：
11.1
通讯作者：
WILLIAMSON, JR
中科院分区：
文献类型：
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作者：
PUGLISI, JD;CHEN, L;WILLIAMSON, JR
人类免疫缺陷病毒 Tat 蛋白与 RNA 茎环结构 (TAR) 特异性结合，该结构包含由三核苷酸凸起分隔的两个螺旋茎区域。 Tat 碱性区域内的单个精氨酸介导与 TAR 的特异性结合，并且作为游离氨基酸的精氨酸也与 TAR 特异性结合。我们之前提出了一个模型，其中精氨酸胍基团与鸟苷 26 (G26) 和一对磷酸盐的相互作用通过在凸起中的 U23 和上螺旋中的 A27.U38 之间形成碱基三重来稳定。在这里，我们通过 NMR 光谱表明，碱基三元组的形成对于精氨酸与 TAR 的结合至关重要。无法形成碱基三元组或去除鸟嘌呤接触的 TAR 突变体不会特异性结合精氨酸。这些突变体还表现出 Tat 的反式激活减少。设计用于在 C23 和 G27.C38 之间形成同形碱基三重的三重突变体结合精氨酸并采用与野生型 TAR 相同的构象。这些结果证明了 RNA 结构对于精氨酸结合的重要性，并进一步证明了精氨酸和 Tat 结合之间的直接对应关系。
The human immunodeficiency virus Tat protein binds specifically to an RNA stem-loop structure (TAR) that contains two helical stem regions separated by a three-nucleotide bulge. A single arginine within the basic region of Tat mediates specific binding to TAR, and arginine as the free amino acid also binds specifically to TAR. We have previously proposed a model in which interaction of the arginine guanidinium group with guanosine-26 (G26) and with a pair of phosphates is stabilized by formation of a base triple between U23 in the bulge and A27.U38 in the upper helix. Here we show by NMR spectroscopy that formation of the base triple is critical for arginine binding to TAR. Mutants of TAR that cannot form the base triple or that remove the guanine contact do not bind arginine specifically. These mutants also showed reduced transactivation by Tat. A triple mutant designed to form an isomorphous base triple between C23 and G27.C38 binds arginine and adopts the same conformation as wild-type TAR. These results demonstrate the importance of RNA structure for arginine binding and further demonstrate the direct correspondence between arginine and Tat binding.