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

新型 PIN_5 结构域型 RNase P 的结构、功能和进化

• 批准号: 409514568
• 负责人: Professor Dr. Roland K. Hartmann
• 金额: --
• 依托单位: Institut für Pharmazeutische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/409514568?language=en
• 关键词: Structure; function; evolution; novel; PIN_5

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.

tRNA 5 '端成熟核酸内切酶RNase P是生命所有领域中的必需酶。RNase P的结构多样性是独一无二的。一类是核糖核蛋白（RNP）复合物，由催化RNA亚基和不同数量（1至10）的蛋白质亚基组成。另一方面，一种缺乏RNA亚基的纯蛋白（或蛋白质）RNase P（PRORP）被发现在真核生物中广泛存在。直到最近，假设细菌中的所有RNase P酶都由催化RNA亚基（约400 nt，由rnpB基因编码）组成，其需要约14 kDa的小蛋白辅因子（由rnpA基因编码）用于体内功能。然而，在一组超嗜热细菌（Aquificaceae），包括Aquifex aeolicus，所有尝试，以确定rnpA和rnpB基因测序的基因组仍然失败了近20年。然而，最近我们成功地在A.风之子这种蛋白质被称为Aq_880，是一种23 kDa的多肽，代表了迄今为止鉴定的最小形式的RNA酶P，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结合结构域。我们发现，Aq_880形成同源寡聚体，并且能够通过灭活更复杂和更大的内源核糖核蛋白RNase P来拯救大肠杆菌和酿酒酵母菌株的生长。在许多细菌和一些细菌中鉴定出Aquifex RNase P（HARP）的同源物，其中所有细菌和大多数细菌也编码基于RNA的RNase P;来自同一细菌或古细菌的两种RNase P形式的活性可以在两种选定的情况下得到验证。生物信息学分析表明，A. aeolicus和相关的Aquificaceae可能通过从古细菌的水平基因转移获得HARP。拟开展的活动的目标是：（i）利用突变和化学发生学方法、RNA结合测定、酶动力学和E. coliRNaseP突变株，（ii）通过一种多层实验策略获得HARP的高分辨率结构，该策略将X射线晶体学、NMR光谱学或冷冻电子显微镜作为选择，（iii）使用co-IP和下拉方法鉴定A.风生菌核糖核酸酶P质谱法（相关蛋白）和RNA-Seq（相关RNA），（iv）在选定的古细菌生物体中HARP的遗传和生物化学分析，以确定古细菌HARP的功能，和（v）在选择的编码两种形式的RNase P的细菌中，对基于RNA的RNase P和HARP进行功能性体外和体内分析，以阐明HARP酶是否可能在进化上是“即将取代这些生物体中古老的RNA酶。