Williams K P, Imahori H, Fujimoto D N, Inoue T
Salk Institute for Biological Studies, San Diego, CA 92037.
Nucleic Acids Res. 1994 Jun 11;22(11):2003-9. doi: 10.1093/nar/22.11.2003.
P5abc is an RNA structure within the self-splicing Tetrahymena group I intron that provides an activation function to the remainder of the ribozyme, either when present in cis or when added in trans. This 69-nucleotide activator domain was replaced with randomized sequence of 20 or 40 nt in length, and individuals among these pools with sequences that could functionally replace P5abc were selected. The basis of selection was a reaction in which two separate halves of the ribozyme became joined; selection was completed by reverse transcription and the polymerase chain reaction, using primers with sequence from either side of the ligation junction. Selectant sequences fell into three families that appear unrelated to P5abc; for example they lack the A-rich bulge thought to be a important feature of P5abc. Thus, rather than defining some consensus sequence for activator domains, this result reveals a certain tolerance in the ribozyme in its ability to derive activation function from diverse sequence types. In the context of splicing precursor RNA, the new sequences supported self-splicing, but failed to activate a related reaction, hydrolysis of the 3' splice site, implying that this region of the intron can differentially control two related reactions.
P5abc是自我剪接的嗜热四膜虫I组内含子中的一种RNA结构，无论其以顺式存在还是以反式添加，都能为核酶的其余部分提供激活功能。这个69个核苷酸的激活结构域被长度为20或40个核苷酸的随机序列所取代，然后从这些文库中筛选出具有能够在功能上替代P5abc序列的个体。筛选的依据是一种反应，在该反应中核酶的两个独立部分连接在一起；通过逆转录和聚合酶链反应完成筛选，使用来自连接位点两侧序列的引物。筛选出的序列分为三个家族，它们似乎与P5abc无关；例如，它们缺乏被认为是P5abc重要特征的富含A的凸起。因此，该结果并未定义激活结构域的某些共有序列，而是揭示了核酶在从不同序列类型获得激活功能的能力方面具有一定的耐受性。在剪接前体RNA的情况下，新序列支持自我剪接，但未能激活相关反应，即3'剪接位点的水解，这意味着内含子的该区域可以差异地控制两个相关反应。
