Structure, function and evolution of the novel PIN_5 domain-type RNases P

TRNA5‘端成熟内切酶RNaseP是生命各个领域中必不可少的酶。RNaseP的结构多样性是独一无二的。一组是核糖核蛋白(RNP)复合体，由一个催化RNA亚基和不同数量(1至10个)的蛋白质亚基组成。另一方面，一种仅含蛋白质(或含蛋白质)的核糖核酸酶P(PRORP)在真核生物中广泛存在，它缺少一个RNA亚基。直到最近，人们一直认为细菌中的所有RNaseP酶都是由一个催化RNA亚基(~400nt，由rnpB基因编码)组成的，该亚基需要一个~14 kDa的小蛋白辅因子(由rnpA基因编码)才能在体内发挥作用。然而，在包括Aquifex aeolicus在内的一组高温嗜热菌中，近20年来，所有从测序基因组中识别rnpA和rnpB基因的尝试都没有成功。然而，最近我们成功地鉴定出了一种新的仅含蛋白质的RNaseP。该蛋白命名为AQ_880，是一种23 kDa的多肽，代表了迄今发现的最小形式的RNaseP，由PIN-5亚型的金属核酸酶结构域组成，但缺乏任何可识别的RNA结合域。我们发现AQ_880形成同源低聚物，能够通过灭活更复杂和更大的内源核糖核蛋白RNaseP来挽救大肠杆菌和酿酒酵母菌株的生长。在许多古生菌和一些细菌中发现了Aquifex RNaseP(HARP)的同源物，其中所有古生菌和大多数细菌也编码基于RNA的RNaseP；来自同一细菌或古生子的两种RNase P形式的活性可以在两个选定的案例中得到验证。生物信息学分析表明，凤眼莲和相关的水生植物很可能是通过从古生体水平转移基因而获得HARP的。拟议活动的目标是：(I)通过突变和化学遗传学方法、RNA结合分析、酶动力学和互补分析，了解HARPS对RNA底物的识别和潜在的切割机制；(Ii)通过结合X射线结晶学、核磁共振光谱或冷冻电子显微镜的多层实验策略，获得HARP的高分辨率结构；(Iii)使用共IP和下拉方法，通过质谱分析(相关蛋白质)和RNA-Seq(相关RNAs)来鉴定Apeolicus RNase P的细胞相互作用伙伴，(Iv)对选定的古生物中的HARP进行遗传和生化分析，以确定古生代HARP的功能，以及(V)对编码这两种RNaseP的选定细菌中基于RNA的RNaseP和HARP进行体外和体内功能分析，以揭示HARP酶是否在进化上“即将”取代/取代这些生物中古老的基于RNA的酶这一有趣的问题。

The tRNA 5'-end maturation endonuclease RNase P is an essential enzyme in all domains of life. The architectural diversity of RNase P is unique. One group are ribonucleoprotein (RNP) complexes consisting of a catalytic RNA subunit and a varying number (1 to 10) of protein subunits. On the other hand, a form of protein-only (or proteinaceous) RNase P (PRORP), lacking an RNA subunit, was found to be widespread among Eukarya. Until recently it was assumed that all RNase P enzymes in Bacteria are composed of a catalytic RNA subunit (~400 nt, encoded by the rnpB gene) that requires a small protein cofactor of ~14 kDa (encoded by the rnpA gene) for in vivo function. However, in a group of hyperthermophilc Bacteria (Aquificaceae) including Aquifex aeolicus, all attempts to identify rnpA and rnpB genes in sequenced genomes remained unsuccessful for almost 20 years. However, very recently we succeeded in identifying a novel type of protein-only RNase P in A. aeolicus. The protein, termed Aq_880, is a 23-kDa polypeptide that represents the smallest form of RNase P identified so far, consisting of a metallonuclease domain of the PIN-5 subtype but lacking any recognizable RNA binding domain. We showed that Aq_880 forms homooligomers and is able to rescue the growth of Escherichia coli and Saccharomyces cerevisiae strains with inactivations of their more complex and larger endogenous ribonucleoprotein RNase P. Homologs of Aquifex RNase P (HARP) were identified in many Archaea and some Bacteria, of which all Archaea and most Bacteria also encode an RNA-based RNase P; activity of both RNase P forms from the same bacterium or archaeon could be verified in two selected cases. Bioinformatic analyses suggest that A. aeolicus and related Aquificaceae likely acquired HARP by horizontal gene transfer from an archaeon. The goals of the proposed activity are: (i) understanding RNA substrate recognition by HARPs and the underlying cleavage mechanism using mutational and chemogenetic approaches, RNA binding assays, enzyme kinetics and complementation analysis in an E. coli RNase P mutant strain, (ii) obtaining high resolution structures of HARPs through a multilayered experimental strategy that incorporates X-ray crystallography, NMR spectroscopy or cryo-electron microscopy as options, (iii) using co-IP and pull-down approaches to identify cellular interaction partners of A. aeolicus RNase P via mass spectrometry (associated proteins) and RNA-Seq (associated RNAs), (iv) genetic and biochemical analyses of HARPs in selected archaeal organisms to determine the function of archael HARPs, and (v) functional in vitro and in vivo analyses of RNA-based RNase P and HARP in selected Bacteria that encode both forms of RNase P to shed light on the fascinating question if the HARP enzyme may evolutionarily be “on the verge of” replacing/displacing the ancient RNA-based enzyme in these organisms.