STRUCTURE AND STABILITY OF RNA PSEUDOKNOTS

这份提案描述了我们针对以下目标的系统方法 理解结构、稳定性和功能之间的关系 在一个结构上相关的RNA假结家族中。伪结点是一种 在自然界中广泛存在的RNA结构的元素，并发挥着 在调节多种生物功能方面起着不可或缺的作用。这个 研究计划将重点放在自动调节内的RNA假结上 噬菌体T2、T4和T6的32mRNAs基因及其移码 调节SRV慢病毒Gag-polmRNA中的伪结点 (猿猴艾滋病)。初步证据有力地表明， 噬菌体和SRV伪结点是一种 结构上相关的RNA伪结的单个大家族，其成员 它们在功能上有所不同。具体地说，以下实验是 建议： (1)噬菌体T2基因32自身调节的结构 伪结与猴逆转录病毒(SRV)Gag-Poll移码导向 伪结将在高分辨率下使用多维确定 核磁共振方法。由于伪节点的功能是发散的，因此 对它们的结构进行比较将有助于了解哪些是 伪结族的共同特征对于泛函是保守的 而不是结构性原因。(2)噬菌体的RNA伪结点 T4和T2/T6基因32个非编码区将作为模型系统 了解结构上被认为的范式的稳定性 这类分子中。一套系统性的基数变化将是 引入终端和内部区域的T4编码 伪结，并确定物理属性是如何被扰动的 相对于野生型RNA。将使用以下方法测量物理属性 紫外线吸收熔化，化学和核糖核酸酶裂解和 亚胺质子核磁共振测量随温度和温度的变化 解决条件。核苷酸变化将由广泛的 由模型派生的最近邻热力学数据库 预测核苷酸作用的寡核苷酸 改变是可能的。

This proposal describes our systematic approach directed toward understanding the relationships between structure, stability and function in a family of structurally related RNA pseudoknots. Pseudoknots are an element of RNA structure that are widespread in nature, and play an integral role in regulating a variety of biological functions. The research plan will focus on RNA pseudoknots within the autoregulatory gene 32 mRNAs of bacteriophages T2, T4 and T6, and the frameshift- regulating pseudoknot within the gag-pol mRNA of the SRV lentivirus (simian AIDS). Preliminary evidence strongly suggests that the bacteriophage and the SRV pseudoknots are representative members of a single large family of structurally related RNA pseudoknots, the members of which differ in function. Specifically the following experiments are proposed: (1) The structures of the bacteriophage T2 gene 32 autoregulatory pseudoknot and the simian retrovirus (SRV) gag-pol frameshift directing pseudoknot will be determined at high resolution using multidimensional NMR methods. Since the functions of the pseudoknots are divergent, a comparison of their structures will provide insight as to which of the common features of the pseudoknot family are conserved for functional versus structural reasons. (2) The RNA pseudoknots of the bacteriophage T4 and T2/T6 gene 32 non-coding regions will be used as model systems to gain an understanding of the stability of the structually deemed paradigm of this class of molecules. A set of systematic base changes will be introduced into the terminal and internal regions of the T4-encoded pseudoknot and determine how the physical properties are perturbed relative to the wild-type RNA. Physical properties will be measured using ultraviolet absorbance melting, chemical and ribonuclease cleavage and imino proton NMR measurements collected as a function of temperature and solution conditions. Nucleotide changes will be guided by an extensive nearest-neighbor thermodynamic database derived from model oligoribonucleotides from which predictions of the effect of nucleotide changes are possible.