NMR STUDIES OF 15N, 13C LABELED RIBONUCLEOTIDES: HIV

SIR提供了13C15N ASI单元；20G [U-“C，”N]为1个细胞；50g艾滋病毒基因组编码两种蛋白质， Tat和Rev，它们是转录的基本调节因子。雷夫 与称为REV反应元件的特定RNA序列结合 (RRE)，发现于HIV基因组中，调节细胞质 编码该基因的未剪接或单剪接的mRNA的外观 结构蛋白Gag、Pol和env。Rev已经被提议拥有 在调节剪接和将RNA运送到 细胞质。化学和核糖核酸酶保护实验表明 66个核苷酸片段，RRE的结构域II是必需的，并且 足以在体外与REV高亲和力结合 仅域H就足以在中检测到版本响应 活着。RRE中的核心版本绑定元素和关键 碱基或碱基-碱基相互作用最近更加精确 由交互式体外遗传选择以及其他 技巧。长含30-35个核苷酸的RNA寡核苷酸 核心结合元件被证明与REV与野生型结合 亲和力。其他研究表明，富含17个氨基酸的精氨酸 来自REV的多肽与RRE在多个位点结合并特异性地抑制 体外拼接。核心区有一个单一的多肽结合 RRE的要素。我们建议美国多维、多核 核磁共振波谱以阐明RRE的结构特征 在REV识别和绑定中很重要。13C和15N标记 用标记的核糖核酸合成的RNA样品 甲烷营养细菌。这些标记的样品将用于核磁共振 核糖核酸和核糖核酸-Rev多肽复合物的结构研究。 还将研究共识RNA序列的各种衍生品 为了进一步明确修订版的结构要求。

The SIR provided 13C15 N ASI cells; 20g [U- "C, "N]AS 1 Cells; 50g The genome of HIV codes for two proteins, Tat and Rev, which are essential regulators of transcription. Rev binds to specific RNA sequence called the Rev Responsive Element (RRE), which is found in the HIV genome and regulates the cytoplasmic appearance of unspliced or singly spliced mRNAs which encode the structural proteins gag, pol, and env. Rev has been proposed to have a role both in regulation of splicing and in transport of the RNA to the cytoplasm. Chemical and RNAse Protection experiments have shown that a 66 nucleotide fragment, domain II of the RRE, is necessary and sufficient for high affinity binding of Rev in in vitro and that domain H alone is sufficient for a detectable Rev responsiveness in vivo. The core Rev binding element within the RRE and the critical bases or base-base interactions have recently been more precisely defined by an interactive in vitro genetic selection as well as other techniques. RNA oligonucleotides 30-35 nucleotides long containing the core binding element were shown to bind Rev with wild type affinity. Other studies have shown that a 17 amino acid arginine rich peptide from Rev binds RRE at multiple sites and specifically inhibits splicing in vitro. A single peptide specifically binds in the core element of the RRE. We propose the us multidimensional, multinuclear NMR spectroscopy to elucidate the structural features of the RRE that are important in REV recognition and binding. 13C and 15N labeled samples of RNA synthesized using labeled NTPs isolated from the RNA of methanolotrophic bacterial. These labeled samples will be used in NMR studies of the structure of RNA and RNA-Rev peptide complexes. Various derivatives of the consensus RNA sequence will also be studied in order to further define the structural requirements for Rev.