Mechanics of a Reversible Cleavage Reaction of RNA

烟草环斑病毒(STobRV)卫星RNA(STobRV RNA)的重复序列、多聚体的两极都会自动裂解，并特异性地产生359个核苷酸残基(NT)，即“单体”sTobRV RNA。负极性的sTobRV(-)RNA也会自发连接以执行切割的3‘-到-5’APG磷酸二酯。STobRV(-)RNA不符合研究较好的“锤头”结构，而这种结构确实适用于sTobRV(+)RNA。STobRV(-)RNA的切割活性序列分散在分子的两个区域，形成一个天然的核酶系统。E是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磷酸二酯上切割它。E和S之间的两个碱基配对片段已经被鉴定出来。我们建议对E-S复合体的特定区域进行系统的突变以确定关键的NT。该复合体还将经过化学修饰以识别三级相互作用，并设计了一个屏幕来识别补偿失活突变的突变。已经合成或计划了新形式的自裂解序列，以促进拟议的研究。我们希望收集到的信息将使我们能够提出一个切割-连接-复合体的结构模型。这一结果将加深我们对sTobRV RNA复制的理解，并为核酶的设计提供便利。

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.