Isolation of fast purine nucleotide synthase ribozymes
Isolation of fast purine nucleotide synthase ribozymes
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DOI：
10.1021/ja045387a
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发表时间：
2004-12-08
影响因子：
15
通讯作者：
Unrau, PJ
中科院分区：
文献类型：
--
作者：
Lau, MWL;Cadieux, KEC;Unrau, PJ
在这里，我们报道了能够促进从束缚的5-磷酸核糖基1-焦磷酸（PRPP）和6-硫鸟嘌呤（（6S）Gua）合成嘌呤核苷酸（6-硫鸟苷单磷酸）的快速核酶的体外选择。两种最高效的嘌呤合酶的表观效率分别为 284 和 230 M-1 min(-1)，并且均比之前通过类似方法选择的嘧啶核苷酸合酶核酶效率显着更高。有趣的是，虽然两种核酶都表现出良好的底物识别能力，但一种核酶对 6-硫鸟嘌呤没有可检测到的亲和力，而第二种核酶的 K-m 类似于 80 muM，表明这些核酶使用相当不同的底物识别模式。经过 10 轮选择后，从两个高多样性 RNA 库中分离出嘌呤合酶。第一个池包含一个长随机序列区域。第二个池包含随机序列元件，其中散布有先前表征的 4-硫尿苷合酶核酶的诱变螺旋元件。虽然几乎所有从这个有偏差的池群体中分离出的核酶似乎都受益于利用祖细胞的螺旋元件之一，但几乎没有证据表明更复杂的二级结构保存。除了嘧啶合酶之外，嘌呤合酶的发现证明了“RNA 世界”中核苷酸合成的潜力，并为研究小分子 RNA 催化提供了背景。
Here we report the in vitro selection of fast ribozymes capable of promoting the synthesis of a purine nucleotide (6-thioguanosine monophosphate) from tethered 5-phosphoribosyl 1-pyrophosphate (PRPP) and 6-thioguanine ((6S)Gua). The two most proficient purine synthases have apparent efficiencies of 284 and 230 M-1 min(-1) and are both significantly more efficient than pyrimidine nucleotide synthase ribozymes selected previously by a similar approach. Interestingly, while both ribozymes showed good substrate discrimination, one ribozyme had no detectable affinity for 6-thioguanine while the second had a K-m of similar to80 muM, indicating that these ribozymes use considerably different modes of substrate recognition. The purine synthases were isolated after 10 rounds of selection from two high-diversity RNA pools. The first pool contained a long random sequence region. The second pool contained random sequence elements interspersed with the mutagenized helical elements of a previously characterized 4-thiouridine synthase ribozyme. While nearly all of the ribozymes isolated from this biased pool population appeared to have benefited from utilizing one of the progenitor's helical elements, little evidence for more complicated secondary structure preservation was evident. The discovery of purine synthases, in addition to pyrimidine synthases, demonstrates the potential for nucleotide synthesis in an 'RNA World' and provides a context from which to study small molecule RNA catalysis.