Murphy F L, Cech T R
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Biochemistry. 1993 May 25;32(20):5291-300. doi: 10.1021/bi00071a003.
The Tetrahymena thermophila pre-rRNA contains a 413-nucleotide self-splicing group I intron. This intron has been converted into a sequence-specific endonuclease or ribozyme. A 160-nucleotide portion of the ribozyme consisting of both highly conserved sequence elements (P4 and P6) and nonconserved peripheral extensions (P5abc and P6ab) was synthesized as a separate molecule. Solvent-based Fe(II)-EDTA, a probe that monitors higher-order RNA structure, revealed a protection pattern that was a large subset of that observed in the whole ribozyme. Data from dimethyl sulfate modification and partial digestion with nucleases were also consistent with maintenance of the proper secondary and tertiary structure in the shortened RNA molecule. Thus, this 160-nucleotide molecule (P4-P6 RNA) is an independently folding domain of RNA tertiary structure. A series of mutations and deletions were made within the P4-P6 domain to further dissect its tertiary structure. Fe(II)-EDTA and dimethyl sulfate analysis of these mutants revealed that the domain consists of two substructures, a localized subdomain involving the characteristic adenosine-rich bulge in P5a, and a subdomain-stabilized structure involving long-range interactions. Therefore, like some proteins, the intron RNA is modular, containing a separable domain and subdomain of tertiary structure.
嗜热四膜虫前体核糖体RNA包含一个413个核苷酸的自我剪接I组内含子。这个内含子已被转化为一种序列特异性内切核酸酶或核酶。由高度保守的序列元件（P4和P6）以及非保守的外围延伸（P5abc和P6ab）组成的160个核苷酸的核酶部分被合成为一个单独的分子。基于溶剂的Fe(II)-EDTA是一种监测高阶RNA结构的探针，它揭示了一种保护模式，该模式是在整个核酶中观察到的保护模式的一个很大的子集。来自硫酸二甲酯修饰和核酸酶部分消化的数据也与缩短的RNA分子中维持适当的二级和三级结构一致。因此，这个160个核苷酸的分子（P4-P6 RNA）是RNA三级结构的一个独立折叠结构域。在P4-P6结构域内进行了一系列突变和缺失，以进一步剖析其三级结构。对这些突变体的Fe(II)-EDTA和硫酸二甲酯分析表明，该结构域由两个子结构组成，一个局部子结构涉及P5a中富含腺苷的特征性凸起，另一个是由长程相互作用稳定的子结构域。因此，与一些蛋白质一样，内含子RNA是模块化的，包含一个可分离的三级结构结构域和子结构域。
