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

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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.