Directed evolution to understand enzyme function of almost protein independent bacterial RNase P RNA versus largely protein-dependent archaeal RNase P RNA

核糖核酸酶P（RNase P）催化所有生物体和细胞器中的tRNA 5 '端成熟。细菌核糖核酸酶P的RNA亚基（P RNA）在无蛋白辅因子的情况下是体外活性催化剂。在进化过程中，真核生物、细胞器和大多数生物的P RNA完全丧失了这种能力，所有重新设计RNA催化的理性尝试都失败了。因此，为了理解P核酶功能的结构和构象要求，需要定向进化的方法。为了实现这一进化策略，我们计划：（a）选择在进化过程中几乎或完全失去其单独RNA活性的编码P RNA的基因进行序列随机化和诱变，以产生具有序列和结构变异的大分子库;（B）体外选择具有P核酶活性的变体和（c）在细菌互补菌株中可以功能性地替代天然P RNA的变体的体内选择。我们期待着以下问题的答案：（1）我们可以反向进化核酶的活性？(2)特异性P RNA核酶功能在构象灵活性和稳定性方面的要求是什么？(3)将非细菌P RNA转化为细菌样P RNA核酶需要哪些结构变化？(4)在体外和体内选择的功能性RNA变体组之间的序列变异有多广泛？(5)细菌宿主中诱变的RNA变体功能的获得是否模仿自然进化？这些RNA在体外会是有效的核酶吗？

Ribonuclease P (RNase P) catalyzes tRNA 5'-end maturation in all organisms and organelles. The RNA subunit (P RNA) of RNase P from Bacteria is an active catalyst in vitro in the absence of the protein cofactor. P RNAs from eucaryotes, organelles and most Archaea have entirely lost this capacity during evolution, and all rational attempts to re-engineer RNA-alone catalysis have failed. Thus, to understand the structural and conformational requirements for P ribozyme function, approaches of directed evolution are needed. To realize such an evolutionary strategy, we plan: (a) sequence randomization and mutagenesis of selected genes encoding P RNAs which have almost or completely lost their RNA-alone activity during evolution, to generate large pools of molecules with sequence and structure variation; (b) in vitro selection of variants with P ribozyme activity and (c) in vivo selection of variants that can functionally replace the native P RNA in bacterial complementation strains. We expect answers to the following questions: (1) Can we retro-evolve ribozyme activity? (2) What are the requirements for specific P RNA ribozyme function in terms of conformational flexibility and stability? (3) Which structural changes are required to convert a non-bacterial P RNA to a bacterial-like P RNA ribozyme? (4) How extensive is sequence variation among the group of functional RNA variants selected in vitro and in vivo? (5) Does a gain of function of mutagenized RNA variants in the bacterial host mimic natural evolution? Will these RNAs be efficient ribozymes in vitro?