Allain F H, Varani G
MRC Laboratory of Molecular Biology, Cambridge, UK.
Nucleic Acids Res. 1995 Feb 11;23(3):341-50. doi: 10.1093/nar/23.3.341.
The upstream site of cleavage of all group I self-splicing introns is identified by an absolutely conserved U.G base pair. Although a wobble C.A pair can substitute the U.G pair, all other combinations of nucleotides at this position abolish splicing, suggesting that it is an unusual RNA structure, rather than sequence, that is recognized by the catalytic intron core. RNA enzymes are metalloenzymes, and divalent metal ion binding may be an important requirement for splice site recognition and catalysis. The paramagnetic broadening of NMR resonances upon manganese binding at specific sites was used to probe the interaction between divalent metal ions and an oligonucleotide model of a group I intron ribozyme substrate. Unlike previous studies in which only imino proton resonances were monitored, we have used isotopically labelled RNA and a set of complete spectral assignments to identify the location of the divalent metal binding site with much greater detail than previously possible. Two independent metal binding sites were identified for this oligonucleotide. A first metal binding site is located in the major groove of the three consecutive G.C base pairs at the end of double helical stem. A second site is found in the major groove of the RNA double helix in the vicinity of the U.G base pair. These results suggest that metal ion coordination (or a metal bridge) and tertiary interactions identified biochemically, may be used by group I intron ribozymes for substrate recognition.
所有I类自我剪接内含子的切割上游位点由一个绝对保守的U.G碱基对确定。尽管摆动的C.A对可以替代U.G对，但该位置核苷酸的所有其他组合都会消除剪接，这表明被催化内含子核心识别的是一种不同寻常的RNA结构，而非序列。RNA酶是金属酶，二价金属离子结合可能是剪接位点识别和催化的重要条件。通过在特定位点结合锰时NMR共振的顺磁加宽来探测二价金属离子与I类内含子核酶底物的寡核苷酸模型之间的相互作用。与之前仅监测亚氨基质子共振的研究不同，我们使用了同位素标记的RNA和一组完整的光谱归属，比以前更详细地确定了二价金属结合位点的位置。为该寡核苷酸鉴定出了两个独立的金属结合位点。第一个金属结合位点位于双螺旋茎末端三个连续G.C碱基对的大沟中。第二个位点位于U.G碱基对附近的RNA双螺旋大沟中。这些结果表明，生化鉴定出的金属离子配位（或金属桥）和三级相互作用可能被I类内含子核酶用于底物识别。
