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(RNaseP)在所有生物和细胞器中催化tRNA5‘端成熟。细菌核糖核酸酶P的RNA亚单位(P-RNA)在缺乏蛋白质辅因子的情况下，在体外是一个活性催化剂。来自真核生物、细胞器和大多数古生物的P RNA在进化过程中完全失去了这种能力，所有重新设计RNA单独催化的合理尝试都失败了。因此，为了了解P核酶功能的结构和构象要求，需要采用定向进化的方法。为了实现这样的进化策略，我们计划：(A)对编码在进化过程中几乎或完全失去单独RNA活性的P-RNA编码的选定基因进行序列随机化和突变，以产生大量具有序列和结构变化的分子库；(B)体外筛选具有P核酶活性的变体；(C)在体内选择能够在功能上取代细菌互补菌株中天然P RNA的变体。我们期待以下问题的答案：(1)我们能否逆进化核酶活性？(2)就构象灵活性和稳定性而言，对特定的P-RNA核酶功能的要求是什么？(3)将非细菌的P-RNA转变为类似细菌的P-RNA核酶需要哪些结构变化？(4)在体外和体内选择的一组功能RNA变体之间的序列差异有多大？(5)在细菌宿主中突变的RNA变体的功能获得是否模拟了自然进化？这些RNA在体外会成为有效的核酶吗？

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?