Pan J, Deras M L, Woodson S A
Department of Chemistry and Biochemistry, University of Maryland, MD 20904-2021, USA.
J Mol Biol. 2000 Feb 11;296(1):133-44. doi: 10.1006/jmbi.1999.3439.
Folding of the Tetrahymena ribozyme under physiological conditions in vitro is limited by slow conversion of long-lived intermediates to the active structure. These intermediates arise because the most stable domain of the ribozyme folds 10-50 times more rapidly than the core region containing helix P3. Native gel electrophoresis and time-resolved X-ray-dependent hydroxyl radical cleavage revealed that mutations that weaken peripheral interactions between domains accelerated folding fivefold, while a point mutation that stabilizes P3 enabled 80 % of the mutant RNA to reach the native conformation within 30 seconds at 22 degrees C. The P3 mutation increased the folding rate of the catalytic core as much as 50-fold, so that both domains of the ribozyme were formed at approximately the same rate. The results show that the ribozyme folds rapidly without significantly populating metastable intermediates when native interactions in the ribozyme core are stabilized relative to peripheral structural elements.
在体外生理条件下，四膜虫核酶的折叠受到长寿命中间体向活性结构缓慢转化的限制。这些中间体的出现是因为核酶最稳定的结构域折叠速度比包含螺旋P3的核心区域快10到50倍。天然凝胶电泳和时间分辨X射线依赖性羟基自由基切割表明，削弱结构域之间外周相互作用的突变使折叠速度加快了五倍，而一个稳定P3的点突变使80%的突变RNA在22摄氏度下30秒内达到天然构象。P3突变使催化核心的折叠速度提高了多达50倍，从而使核酶的两个结构域以大致相同的速度形成。结果表明，当核酶核心中的天然相互作用相对于外周结构元件得到稳定时，核酶能够快速折叠，而不会大量形成亚稳态中间体。
