In vitro evolution of coenzyme-independent variants from the glmS ribozyme structural scaffold.
In vitro evolution of coenzyme-independent variants from the glmS ribozyme structural scaffold.
复制标题
DOI：
10.1016/j.ymeth.2016.04.027
复制
发表时间：
2016-08-15
期刊：
影响因子：
--
通讯作者：
Ferré-D'Amaré AR
中科院分区：
文献类型：
--
作者：
Lau MW;Ferré-D'Amaré AR
在已知的切割RNA序列的天然核酶中是独一无二的--具体地说，GLMS核酶-核糖开关使用一个小分子，氨基葡萄糖-6-磷酸(GlcN6P)作为催化辅因子。体外筛选用于寻找该核酶的辅酶非依赖性变异体。除了揭示核酶的催化机制外，这些变体还可能类似于现代GlcN6P调控的核酶-核糖开关的进化祖先。构建了一个突变库，以保存定义核酶的三重假结全局折叠的二级结构元素。依赖于硫醇-汞亲和层析分离活性和非活性序列的严格选择方案最终产生了三个突变体，其切割速率超过3min−1，仅需要二价阳离子来进行活性。突变分析表明，变异体向野生型序列的点回复足以部分恢复对GlcN6P的依赖，这表明辅酶依赖可以很容易地被采用GLMS核酶折叠的RNA获得。本文详细介绍了进行这一选择实验的方法。
Uniquely among known natural ribozymes that cleave RNA sequence-specifically, the glmS ribozyme-riboswitch employs a small molecule, glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor. In vitro selection was employed to search for coenzyme-independent variants of this ribozyme. In addition to shedding light on the catalytic mechanism of the ribozyme, such variants could resemble the evolutionary ancestors of the modern, GlcN6P-regulated ribozyme-riboswitch. A mutant pool was constructed such that the secondary structure elements, which define the triply-pseudoknotted global fold of the ribozyme, was preserved. A stringent selection scheme that relies on thiol-mercury affinity chromatography for separating active and inactive sequences ultimately yielded a triple mutant with a cleavage rate exceeding 3 min−1 that only requires divalent cations for activity. Mutational analysis demonstrated that a point reversion of the variant toward the wild-type sequence was sufficient to partially restore GlcN6P-dependence, suggesting that coenzyme dependence can be readily be acquired by RNAs that adopt the glmS ribozyme fold. The methods employed to perform this selection experiment are described in detail in this review.