RNA oligomers of length 40–60 nt can self-assemble into covalent versions of the Azoarcus group I intron ribozyme. This process requires a series of recombination reactions in which the internal guide sequence of a nascent catalytic complex makes specific interactions with a complement triplet, CAU, in the oligomers. However, if the CAU were mutated, promiscuous self-assembly may be possible, lessening the dependence on a particular set of oligomer sequences. Here, we assayed whether oligomers containing mutations in the CAU triplet could still self-construct Azoarcus ribozymes. The mutations CAC, CAG, CUU and GAU all inhibited self-assembly to some degree, but did not block it completely in 100 mM MgCl2. Oligomers containing the CAC mutation retained the most self-assembly activity, while those containing GAU retained the least, indicating that mutations more 5′ in this triplet are the most deleterious. Self-assembly systems containing additional mutant locations were progressively less functional. Analyses of properly self-assembled ribozymes revealed that, of two recombination mechanisms possible for self-assembly, termed ‘tF2’ and ‘R2F2’, the simpler one-step ‘tF2’ mechanism is utilized when mutations exist. These data suggest that self-assembling systems are more facile than previously believed, and have relevance to the origin of complex ribozymes during the RNA World.
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机译：长度为40–60 nt的RNA低聚物可以自组装成Azoarcus I类内含子核酶的共价形式。该过程需要一系列重组反应，其中新生催化复合物的内部引导序列与寡聚体中的补体三联体CAU发生特异性相互作用。但是，如果CAU发生突变，混杂的自组装是可能的，从而减少了对特定寡聚物序列集的依赖性。在这里，我们分析了在CAU三联体中含有突变的寡聚体是否仍然可以自我构建牛带固氮菌核酶。 CAC，CAG，CUU和GAU突变均在一定程度上抑制了自组装，但并未在100 mM MgCl2中完全阻断其自组装。含有CAC突变的寡聚体保留了最多的自组装活性，而含有GAU的寡聚体保留了最少的自组装活性，表明该三联体中5'处的突变最有害。包含其他突变体位置的自组装系统的功能逐渐降低。对正确自我组装的核酶的分析表明，在可能用于自我组装的两种重组机制（称为“ tF2”和“ R2F2”）中，当存在突变时，使用了更简单的一步“ tF2”机制。这些数据表明，自组装系统比以前认为的要容易得多，并且与RNA世界期间复杂核酶的起源有关。
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