Abstract:RNase P is primarily responsible for the 5΄ maturation of transfer RNAs (tRNAs) in all domains of life. Archaeal RNase P is a ribonucleoprotein made up of one catalytic RNA and five protein cofactors including L7Ae, which is known to bind the kink-turn (K-turn), an RNA structural element that causes axial bending. However, the number and location of K-turns in archaeal RNase P RNAs (RPRs) are unclear. As part of an integrated approach, we used native mass spectrometry to assess the number of L7Ae copies that b… Show more
“…Phylogenetic and secondary structural analyses revealed that an extension with a K-turn beyond stem P12 is a conserved feature in most archaeal RNase P RNAs (Supplementary Fig. 7) 30,37,38,43,44 .Fig. 3Structure of the M. jannaschii RPR.…”
Ribonuclease P (RNase P) is an essential ribozyme responsible for tRNA 5′ maturation. Here we report the cryo-EM structures of
Methanocaldococcus jannaschii
(
Mja
) RNase P holoenzyme alone and in complex with a tRNA substrate at resolutions of 4.6 Å and 4.3 Å, respectively. The structures reveal that the subunits of
Mja
RNase P are strung together to organize the holoenzyme in a dimeric conformation required for efficient catalysis. The structures also show that archaeal RNase P is a functional chimera of bacterial and eukaryal RNase Ps that possesses bacterial-like two RNA-based anchors and a eukaryal-like protein-aided stabilization mechanism. The 3′-RCCA sequence of tRNA, which is a key recognition element for bacterial RNase P, is dispensable for tRNA recognition by
Mja
RNase P. The overall organization of
Mja
RNase P, particularly within the active site, is similar to those of bacterial and eukaryal RNase Ps, suggesting a universal catalytic mechanism for all RNase Ps.
“…Phylogenetic and secondary structural analyses revealed that an extension with a K-turn beyond stem P12 is a conserved feature in most archaeal RNase P RNAs (Supplementary Fig. 7) 30,37,38,43,44 .Fig. 3Structure of the M. jannaschii RPR.…”
Ribonuclease P (RNase P) is an essential ribozyme responsible for tRNA 5′ maturation. Here we report the cryo-EM structures of
Methanocaldococcus jannaschii
(
Mja
) RNase P holoenzyme alone and in complex with a tRNA substrate at resolutions of 4.6 Å and 4.3 Å, respectively. The structures reveal that the subunits of
Mja
RNase P are strung together to organize the holoenzyme in a dimeric conformation required for efficient catalysis. The structures also show that archaeal RNase P is a functional chimera of bacterial and eukaryal RNase Ps that possesses bacterial-like two RNA-based anchors and a eukaryal-like protein-aided stabilization mechanism. The 3′-RCCA sequence of tRNA, which is a key recognition element for bacterial RNase P, is dispensable for tRNA recognition by
Mja
RNase P. The overall organization of
Mja
RNase P, particularly within the active site, is similar to those of bacterial and eukaryal RNase Ps, suggesting a universal catalytic mechanism for all RNase Ps.
“…The Rpp21/Rpp29 pair was shown to contribute to the pre-tRNA substrate affinity ( 15 ). Additionally, ribosomal protein L7Ae is a part of archaeal RNase P and was shown to bind to kink-turn motifs in RNase P RNA and contribute to the activity and temperature stability of the archaeal RNP ( 17 , 18 ).…”
RNase P is a ubiquitous site-specific endoribonuclease primarily responsible for the maturation of tRNA. Throughout the three domains of life, the canonical form of RNase P is a ribonucleoprotein (RNP) built around a catalytic RNA. The core RNA is well conserved from bacteria to eukaryotes, whereas the protein parts vary significantly. The most complex and the least understood form of RNase P is found in eukaryotes, where multiple essential proteins playing largely unknown roles constitute the bulk of the enzyme. Eukaryotic RNase P was considered intractable to in vitro reconstitution, mostly due to insolubility of its protein components, which hindered its studies. We have developed a robust approach to the in vitro reconstitution of Saccharomyces cerevisiae RNase P RNPs and used it to analyze the interplay and roles of RNase P components. The results eliminate the major obstacle to biochemical and structural studies of eukaryotic RNase P, identify components required for the activation of the catalytic RNA, reveal roles of proteins in the enzyme stability, localize proteins on RNase P RNA, and demonstrate the interdependence of the binding of RNase P protein modules to the core RNA.
“…Diversity of the ribonucleoprotein variants of RNase P, and inventory of the multifunctional protein subunits of archaeal/eukaryal RNase P. Overlaid on the secondary structure of the respective RNA subunit are the different RNase P protein subunits. The approximate placement of the protein subunits is based on data reported elsewhere (Tsai et al 2006;Pulukkunat and Gopalan 2008;Xu et al 2009;Chen et al 2010;Reiter et al 2010;Hipp et al 2012;Khanova et al 2012;Fagerlund et al 2015;Lai et al 2017). For subunits that have been demonstrated to have roles other than RNase P catalysis, these are indicated using one-or two-letter codes (see key for explanation of the codes).…”
RNase P catalyzes 5'-maturation of tRNAs in all three domains of life. This primary function is accomplished by either a ribozyme-centered ribonucleoprotein (RNP) or a protein-only variant (with one to three polypeptides). The large, multicomponent archaeal and eukaryotic RNase P RNPs appear disproportionate to the simplicity of their role in tRNA 5'-maturation, prompting the question of why the seemingly gratuitously complex RNP forms of RNase P were not replaced with simpler protein counterparts. Here, motivated by growing evidence, we consider the hypothesis that the large RNase P RNP was retained as a direct consequence of multiple roles played by its components in processes that are not related to the canonical RNase P function.
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