DIVALENT METAL-ION BINDING TO A CONSERVED WOBBLE PAIR DEFINING THE UPSTREAM SITE OF CLEAVAGE OF GROUP-I SELF-SPLICING INTRONS
DIVALENT METAL-ION BINDING TO A CONSERVED WOBBLE PAIR DEFINING THE UPSTREAM SITE OF CLEAVAGE OF GROUP-I SELF-SPLICING INTRONS
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DOI：
10.1093/nar/23.3.341
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发表时间：
1995-02-11
影响因子：
14.9
通讯作者：
VARANI, G
中科院分区：
文献类型：
--
作者：
ALLAIN, FHT;VARANI, G
所有I组自剪接内含子的切割上游位点由绝对保守的U.G碱基对鉴定。虽然摆动的C. A对可以取代U.G对，但在这个位置上的所有其他核苷酸组合都取消了剪接，这表明它是一种不寻常的RNA结构，而不是序列，被催化内含子核心识别。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要求。在特定位点锰结合后的NMR共振的顺磁加宽被用来探测二价金属离子和I组内含子核酶底物的寡核苷酸模型之间的相互作用。与以前的研究中，只有亚氨基质子共振进行监测，我们使用同位素标记的RNA和一组完整的光谱分配，以确定的位置的二价金属结合位点的细节比以前可能的。两个独立的金属结合位点被确定为这种寡核苷酸。第一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RNA双螺旋的大沟中U.G碱基对附近。这些结果表明，金属离子配位（或金属桥）和三级相互作用的生化鉴定，可用于第一组内含子核酶的底物识别。
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.