Mechanics of a Reversible Cleavage Reaction of RNA

烟草环斑病毒卫星RNA （sTobRV RNA）的重复序列、多聚体形式的两个极性都能自裂解和特异性切割，生成359个核苷酸残基（nt）的“单体”sTobRV RNA。负极性的sTobRV (-)RNA也会自发连接，进行3‘到5’ apg磷酸二酯的裂解。sTobRV (-)RNA不符合已被充分研究的“锤头”结构，而这种结构适用于sTobRV (+)RNA。sTobRV (-)RNA的裂解活性序列分散在分子的两个区域，形成天然的“核酶”系统。E，一个与这两个区域中的一个相对应的寡核苷酸，作用于另一个区域S，并在ApG磷酸二酯处切割它。E和S之间的碱基配对的两个片段先前已被确定。我们建议对E-S复合物的特定区域进行系统诱变以识别关键nt。该复合物也将受到化学修饰以识别三级相互作用，并且已经设计了一个筛选来识别补偿失活突变的突变。新形式的自切割序列已被合成，或计划，以促进提出的研究。我们期望收集到的信息将使我们能够提出解理-结扎-复合物的结构模型。该结果将提高我们对sTobRV RNA复制的理解，并有助于“核酶”的设计。

Both polarities of the repetitive-sequence, multimeric forms of the satellite RNA of tobacco ringspot virus (sTobRV RNA) cleave autolytically and specifically to generate 359 nucleotide residue (nt), "monomeric" sTobRV RNA. The negative polarity sTobRV (-)RNA also spontaneously ligates to perform the cleaved 3'-to-5'ApG phosphodiester. sTobRV (-)RNA does not conform to the well-studied "hammerhead" structure, which does apply to sTobRV (+)RNA. The cleavage-active sequences of sTobRV (-)RNA are dispersed in two regions of the molecule, forming a natural "ribozyme" system. E, an oligoribonucleotide corresponding to one of these two regions, acted on the other, S, and cleaved it at the ApG phosphodiester. Two segments of base pairing between E and S previously have been identified. We propose a systematic mutagenesis of specific regions of the E-S complex to identify critical nt. The complex also will be subjected to chemical modification to identify tertiary interactions, and a screen has been designed to identify mutations which compensate for inactivating mutations. Novel forms of the self-cleaving sequence have been synthesized, or planned, to facilitate the proposed investigations. We expect that the information gathered will allow us to propose a structural model for the cleavage- ligation-complex. The results will improve our understanding of sTobRV RNA replication and facilitate "ribozyme" design.