In vitro selection for altered divalent metal specificity in the RNase P RNA
In vitro selection for altered divalent metal specificity in the RNase P RNA
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DOI：
10.1073/pnas.94.26.14355
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发表时间：
1997-12-23
影响因子：
11.1
通讯作者：
Pace, NR
中科院分区：
文献类型：
--
作者：
Frank, DN;Pace, NR
核酶RNase P绝对需要二价金属离子才能发挥催化作用。多个Mg2+离子有助于RNase P的最佳催化效率，很可能是核酶的三级结构在活性位点为这些离子形成了一个特定的金属结合袋。为了鉴定有助于催化金属结合位点的碱基，我们使用体外选择分离大肠杆菌RNase P RNA的变体，这些变体对二价金属的特异性发生了改变。在Ca2+中活性增加的RNase P RNA变异在18代的选择中富集，在Ca2+存在下进行催化，这通常相对于Mg2+不利。尽管在is代克隆中发现了广泛的突变，但所有克隆中只有一个共同的突变：RNase p的第70位（大肠杆菌编号）的胞嘧啶到尿嘧啶的转变。在野生型背景下对C70U点突变体的分析证实，第70位碱基的身份是Ca2+选择性的唯一决定因素。值得注意的是，C70位于系统发育保守的J3/4-P4-J2/4区域，先前与Mg2+结合有关。我们发现单个碱基的改变足以改变RNase P的金属偏好，这进一步证明了J3/4-P4-J2/4结构域构成了核酶活性位点的一部分。
The ribozyme RNase P absolutely requires divalent metal ions for catalytic function. Multiple Mg2+ ions contribute to the optimal catalytic efficiency of RNase P, and it is likely that the tertiary structure of the ribozyme forms a specific metal-binding pocket for these ions within the active-site. To identify base moieties that contribute to catalytic metal-binding sites, we have used in vitro selection to isolate variants of the Escherichia coli RNase P RNA with altered specificities for divalent metal. RNase P RNA variants with increased activity in Ca2+ were enriched over 18 generations of selection for catalysis in the presence of Ca2+, which is normally disfavored relative to Mg2+. Although a wide spectrum of mutations was found in the generation-IS clones, only a single paint mutation was common to all clones: a cytosine-to-uracil transition at position 70 (E. coli numbering) of RNase P. Analysis of the C70U point mutant in a wild-type background confirmed that the identity of the base at position 70 is the sole determinant of Ca2+ selectivity. It is noteworthy that C70 lies within the phylogenetically well conserved J3/4-P4-J2/4 region, previously implicated in Mg2+ binding. Our finding that a single base change is sufficient to alter the metal preference of RNase P is further evidence that the J3/4-P4-J2/4 domain forms a portion of the ribozyme's active site.