CATALYTIC RNA

来自几个植物卫星RNA的某些RNA序列表现出自溶 导致特定部位切割的酯交换反应 反应(箭头)。这些RNA中常见的小的共同序列可以 由两个(或三个)片段组成，由三个螺旋组成 (其中一个或多个可以在发夹环中终止) 被称为锤头鲨。有九个保守的非螺旋核苷 残基(和一个或两个碱基对)似乎对 观察到了卵裂活动。 这个项目将决定具体基地和 碳水化合物官能团可用在十三个保守的 核苷酸残基，其中一些将参与关键的碱基间 氢键、碱基堆积或镁相互作用 观察到的自催化功能。关键官能团将 通过制备一系列“缺失修饰”核酶进行鉴定 具有位于碱基上的特定官能团或 保守核苷酸的碳水化合物残基具有选择性 已删除。这项研究的目标官能团包括嘌呤 和嘧啶环氮以及外环氨基和羰基 组。修饰后的碎片将通过化学合成来制备。 共14个改性核苷亚磷酰胺将用于 特定于站点的删除修改合并到 核酶或底物片段。到那时，卵裂的相对速度将 在化学计量和催化条件下均可测定。我们 将比较切除核酶/底物结构的效果 (CD、热稳定性和核磁共振)将允许解剖几何图形 并使我们能够提出和活跃的复合体的位置构象。 拟议工作的结果将阐明通过什么进程 某些RNA序列可以采用独特的二级/三级结构来 精确定位特定官能团以结合镁离子和标记化 弯刀状的磷酸二酯键。

Certain RNA sequences from several plant satellite RNAs exhibit autolytic transesterification reactions that result in site-specific cleavage reactions (arrow). The small consensus sequence common in these RNAs can be formed from two (or three) fragments and is composed of three helices (one or more of which can terminate in a hairpin loop) and has been termed a hammerhead. There are nine conserved non-helical nucleoside residues (and one or two base pairs) that appear to be critical for the observed cleavage activity. This project will determine the importance of specific base and carbohydrate functional group available on the thirteen conserved nucleotide residues, some of which will be involved in key interbase hydrogen bonding, base stacking or magnesium interactions necessary for the observed autocatalytic function. The critical functional groups will be identified by preparing a series of "deletion modified" ribozyme complexes in which specific functional groups located on the base or carbohydrate residues of the conserved nucleotides are selectivity deleted. The functional groups targeted in this study include the purine and pyrimidine ring nitrogen as well as the exocyclic amino and carbonyl groups. The modified fragments will be prepared by chemical syntheses. A total of 14 modified nucleoside phosphoramidites will be prepared for site-specific incorporation of the deletion modifications into the ribozyme or substrate fragment. The relative rates of cleavage will then be determined under both stoichiometric and catalytic conditions. We will compare the effects of the excision ribozyme/substrate structure (CD, thermal stability, and NMR) will allow dissection of the geometry and enable us to propose and active site conformation for the complex. The results of the proposed work will shed light on the process by which certain RNA sequences can adopt a unique secondary/tertiary structure to precisely position specific functional groups to bind Mg2+ and labilize the scissle phosphodiester bond.