ARGININE-MEDIATED RNA RECOGNITION

精氨酸介导的 RNA 识别

• 批准号: 2184931
• 负责人: ALAN D FRANKEL
• 金额: $ 19.79万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2184931
• 关键词: DNA; RNA; RNA binding protein; X ray crystallography; arginine; circular dichroism; conformation; mutant; nuclear magnetic resonance spectroscopy; nucleic acid sequence; polymerase chain reaction; protein folding; transfection

RNA-protein interactions are essential for many regulatory and cellular processes, including transcription, RNA splicing, and translation. Although these interactions play crucial roles in the cell, only a modest amount is known about the details of sequence-specific RNA recognition, in part because RNA can fold into complicated tertiary structures. It is clear that both RNA structure and base sequence play important roles in recognition. The study of RNA-protein interactions has been somewhat simplified by the discovery of a few common motifs in RNA-binding proteins. One of these, the arginine-rich motif, is found in the HIV Tat protein. Tat uses a single arginine residue, in the midst of a region of basic amino acids, to specifically recognize the TAR RNA hairpin. Arginine appears to recognize a particular structural feature of TAR, probably simultaneously hydrogen bonding to two phosphates on the backbone and to a base in the major groove. The interaction of arginine with two adjacent phosphates, termed an "arginine fork", provides a possible mechanism for a protein to indirectly read out a base sequence by recognizing a sequence- dependent RNA structure. Other modes of binding have been seen with arginine and the guanosine-binding site of a group 1 intron, and with arginines of tRNA synthetases involved in tRNA recognition. This proposal will use the specific binding of arginine as a tool to probe RNA structure and folding and to define arginine-RNA interactions that may be important in RNA-protein recognition. The finding that short arginine-containing peptides, or even free arginine, can bind specifically to RNAs makes it possible to ask which features of RNA structure cause it to adopt specific arginine-binding conformations. RNA selection methods will be used to identify RNAs from random mixtures that specifically bind arginine. Groups on the RNA bases and backbone that interact with arginine will be mapped by chemical modification and mutagenesis experiments. Possible conformational changes in the RNAs, which have been seen in other RNA-protein interactions, will be examined by circular dichroism and NMR experiments. the role of surrounding charged amino acids within the arginine-rich motif will be determined by mutagenesis in the Tat-TAR system and by structural comparisons of RNAs selected with peptides. Selection experiments will be used to ask whether DNAs can adopt specific arginine-binding conformations and whether these structures are similar to or different than the selected RNAs. RNAs will be prepared to determine the detailed three-dimensional structures of interesting RNAs and arginine-RNA complexes by NMR and X- ray crystallography. It is anticipated that the studies proposed here will shed light on fundamental features of RNA-protein recognition, RNA folding, and RNA structure, and thus be important for understanding mechanisms of cellular regulation.

RNA - 蛋白质相互作用对于许多调节和细胞都是必不可少的 过程，包括转录，RNA剪接和翻译。 尽管这些相互作用在细胞中起着至关重要的作用，但只有一个谦虚 关于序列特异性RNA识别的细节已知数量， 部分原因是RNA可以折叠成复杂的三级结构。 它 清楚地表明，RNA结构和碱基序列都起着重要的作用 在认可中。 RNA - 蛋白质相互作用的研究已经通过 在RNA结合蛋白中发现了一些常见基序。 其中之一 在HIV TAT蛋白中发现了富含精氨酸的基序。 tat使用 在碱性氨基酸的区域中，单精氨酸残基 专门识别焦油RNA发夹。 精氨酸似乎是 识别焦油的特定结构特征，可能同时 氢键在主链上的两个磷酸盐和底座上 主要凹槽。 精氨酸与两个相邻磷酸盐的相互作用， 称为“精氨酸叉”，为蛋白质提供了一种可能的机制 通过识别序列 - 依赖RNA结构。 与 精氨酸和1组内含子的鸟嘌呤结合部位，并带有 与tRNA识别有关的tRNA合酶的精氨酸。 该建议将使用精氨酸的特定结合作为工具 探针RNA结构和折叠并定义精氨酸-RNA相互作用 这在RNA蛋白识别中可能很重要。 发现简短 含精氨酸的肽，甚至是游离精氨酸都可以结合 专门针对RNA可以询问RNA的哪些功能 结构导致它采用特定的精氨酸结合构象。 RNA 选择方法将用于从随机混合物中识别RNA 特异性结合精氨酸。 RNA碱基和骨干的组 与精氨酸相互作用将通过化学修饰和 诱变实验。 RNA可能的构象变化， 在其他RNA - 蛋白质相互作用中已经看到的，将被检查 通过圆形二色性和NMR实验。 周围的角色 富含精氨酸的基序中带电的氨基酸将由 TAT-TAR系统中的诱变和通过RNA的结构比较 选择肽。 选择实验将用于询问 DNA是否可以采用特定的精氨酸结合构象和 这些结构与所选的结构相似还是不同 RNA。 RNA将准备确定详细的三维 NMR和X-的有趣RNA和精氨酸RNA复合物的结构 射线晶体学。 预计在这里提出的研究 会阐明RNA蛋白识别的基本特征RNA 折叠和RNA结构，因此对于理解很重要 细胞调节的机制。