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

SIR提供13 C15 N ASI细胞; 20 g [U-“C，“N]AS 1细胞; 50 g HIV的基因组编码两种蛋白质， 达特和Rev，它们是转录的基本调节因子。 Rev 与称为Rev反应元件的特定RNA序列结合 (RRE)，它存在于HIV基因组中，调节细胞质中的 未剪接或单剪接的mRNA的出现， 结构蛋白gag、pol和env。 已建议Rev 在剪接调节和RNA转运中的作用 细胞质 化学和RNA酶保护实验表明， 一个66个核苷酸的片段，RRE的结构域II，是必需的， 足以在体外高亲和力结合Rev， 单独的结构域H足以在细胞中产生可检测的Rev响应性。 vivo. RRE中的核心Rev约束元素和关键 碱基或碱基间的相互作用最近被更精确地 通过交互式体外遗传选择以及其他 技术. RNA寡核苷酸30-35个核苷酸长，含有 核心结合元件显示与野生型Rev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的肽在多个位点结合RRE，并特异性抑制 体外剪接。 一个单一的肽特异性地结合在核心 RRE的元素。 我们建议美国多层面，多核 NMR光谱以阐明RRE的结构特征， 在REV识别和绑定中很重要。 13 C和15 N标记 使用分离自以下的RNA的标记的NTP合成的RNA样品： 产甲烷细菌 这些标记的样品将用于NMR RNA和RNA-Rev肽复合物的结构研究。 各种衍生物的共识RNA序列也将进行研究 为进一步明确Rev.

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.