Protein-only RNase P function in Escherichia coli: viability, processing defects and differences between PRORP isoenzymes

Gößringer, Markus; Lechner, Marcus; Brillante, Nadia; Weber, Christoph; Rossmanith, Walter; Hartmann, Roland K.

doi:10.1093/nar/gkx405

Cited by 21 publications

(25 citation statements)

References 56 publications

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“…However, even strains harboring the less effectively complementing A. thaliana PRORP1 grew better than any of the Aq_880 strains. The slow growth could be due to problems in expressing sufficient bacterial protein in the eukaryal host, unfavorable subcellular localization, differences in substrate recognition between Aq_880 and yeast nuclear RNase P, or a combination of them leading to retarded biogenesis of mature tRNAs in general, or of a subset of tRNAs as observed for A. thaliana PRORP1 complementation in E. coli (20). Bioinformatic screens identified numerous Aq_880 homologs in Archaea, few in Bacteria (Fig.…”

Section: Resultsmentioning

confidence: 99%

Minimal and RNA-free RNase P in Aquifex aeolicus

Nickel

Wäber

Gößringer

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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RNase P is an essential tRNA-processing enzyme in all domains of life. We identified an unknown type of protein-only RNase P in the hyperthermophilic bacterium Aquifex aeolicus: Without an RNA subunit and the smallest of its kind, the 23-kDa polypeptide comprises a metallonuclease domain only. The protein has RNase P activity in vitro and rescued 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.protein-only RNase P | Aquifex aeolicus | tRNA processing | HARP T he architectural diversity of RNase P enzymes is unique: In Bacteria, Archaea, and in the nuclei and organelles of many Eukarya, RNase P is a complex consisting of a catalytic RNA subunit and a varying number of proteins (one in Bacteria, at least four in Archaea, and up to 10 in Eukarya) (1, 2). A different type of RNase P was discovered more recently in human mitochondria (3) and, subsequently, in land plants and some protists (4, 5). This form, termed proteinaceous or protein-only RNase P (PRORP), lacks any RNA subunit and consists of one or three (animal mitochondria) protein subunit(s); it is found in most branches of the eukaryotic phylogenetic tree (6).Bacterial RNase P enzymes identified so far are composed of a ∼400-nt-long catalytic RNA subunit (encoded by rnpB) and a small protein subunit of ∼14 kDa (encoded by rnpA) (7). However, no rnpA and rnpB genes were identified in the genome of Aquifex aeolicus or other Aquificaceae (8-12). The genetic organization of A. aeolicus tRNAs in tandem clusters and as part of ribosomal operons and the detection of tRNAs with canonical mature 5′-ends in total RNA extracts from A. aeolicus implied the existence of a tRNA 5′-maturation activity (9) that was indeed subsequently detected in cell lysates of A. aeolicus (11, 13). However, to date, the identity and biochemical composition of RNase P in A. aeolicus has remained enigmatic. Results and DiscussionHere, we pursued a classical biochemical approach to identify the RNase P of A. aeolicus. The purification procedure consisted of three consecutive chromatographic steps: anion exchange, hydrophobic interaction, and size exclusion chromatography (AEC, HIC, and SEC, respectively; Fig. 1A and SI Appendix, Figs. S1-S8). RNase P activity was assayed at all purification steps. To identify putative protein components of the enzyme, fractions with low and high RNase P activity from different purification steps were comparatively analyzed by step-gradient SDS/PAGE, and protein bands correlating with activity (Fig. 1B) were subjected to mass spectrometry. An example i...

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Section: Resultsmentioning

confidence: 99%

Minimal and RNA-free RNase P in Aquifex aeolicus

Nickel

Wäber

Gößringer

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Derivatives of plasmid pDG148(S/X) encoding the HARP proteins without and with C‐terminal His‐tag were transformed into E. coli BW for complementation experiments carried out as described previously .…”

Section: Methodsmentioning

confidence: 99%

“…As the eukaryal PRORPs, Aq_880 is also a member of the PIN domain‐like superfamily of metallonucleases, but belonging to a different subgroup (PIN_5 cluster, VapC structural group) . In contrast to the known PRORPs (~60–65 kDa ), Aq_880 is a remarkably small protein (~23 kDa), which shows limited similarity to the metallonuclease domain of PRORPs but lacks their pentatricopeptide repeat (PPR) RNA‐binding and bipartite zinc‐binding domains . Homologs of Aq_880, termed Homologs of Aquifex RNase P (HARPs), were identified in some bacteria and many archaea (see alignment in Fig.…”

Section: Introductionmentioning

confidence: 99%

Homologs of aquifex aeolicus protein‐only RNase P are not the major RNase P activities in the archaea haloferax volcanii and methanosarcina mazei

et al. 2019

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The mature 5′‐ends of tRNAs are generated by RNase P in all domains of life. The ancient form of the enzyme is a ribonucleoprotein consisting of a catalytic RNA and one or more protein subunits. However, in the hyperthermophilic bacterium Aquifex aeolicus and close relatives, RNase P is a protein‐only enzyme consisting of a single type of polypeptide (Aq_880, ~23 kDa). In many archaea, homologs of Aq_880 were identified (termed HARPs for Homologs of Aquifex RNase P) in addition to the RNA‐based RNase P, raising the question about the functions of HARP and the classical RNase P in these archaea. Here we investigated HARPs from two euryarchaeotes, Haloferax volcanii and Methanosarcina mazei. Archaeal strains with HARP gene knockouts showed no growth phenotypes under standard conditions, temperature and salt stress (H. volcanii) or nitrogen deficiency (M. mazei). Recombinant H. volcanii and M. mazei HARPs were basically able to catalyse specific tRNA 5′‐end maturation in vitro. Furthermore, M. mazei HARP was able to rescue growth of an Escherichia coli RNase P depletion strain with comparable efficiency as Aq_880, while H. volcanii HARP was unable to do so. In conclusion, both archaeal HARPs showed the capacity (in at least one functional assay) to act as RNases P. However, the ease to obtain knockouts of the singular HARP genes and the lack of growth phenotypes upon HARP gene deletion contrasts with the findings that the canonical RNase P RNA gene cannot be deleted in H. volcanii, and a knockdown of RNase P RNA in H. volcanii results in severe tRNA processing defects. We conclude that archaeal HARPs do not make a major contribution to global tRNA 5′‐end maturation in archaea, but may well exert a specialised, yet unknown function in (t)RNA metabolism. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1109–1116, 2019

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“…This level of subunit complexity of the archaeal and eukaryotic RNase P RNP variants appears disproportionate for hydrolysis of a single phosphodiester bond in a precursor tRNA, given that four of the five eukaryotic super-groups also possess typical, moderately sized (∼60 kDa) protein-only RNase P (PRORP) having a similar enzymatic activity (Gobert et al 2010;Lechner et al 2015). Moreover, genetic complementation studies in Escherichia coli and Saccharomyces cerevisiae indicate that some of the PRORPs can substitute for the RNase P RNP activity without detrimental effects on growth under laboratory conditions (Weber et al 2014;Gößringer et al 2017). In one genetic background and under high-salt conditions, a strain of S. cerevisiae with a protein-only form (a single polypeptide from Arabidopsis) as the functional RNase P was able to outcompete in growth a wild-type counterpart that uses the 10-subunit RNP (Weber et al 2014).…”

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confidence: 99%

“…The above observations that emphasize the basis for retention of the larger RNP isoforms of RNase P need to be squared with the successful genetic replacement of the RNP with a protein-only form in E. coli and yeast (Weber et al 2014;Gößringer et al 2017). This neutral swap indicates the elasticity to rewire a central housekeeping enzyme, but its biological impact remains to be determined since genetic or environmental factors could engender different organismal robustness outcomes within the framework of this "neutral" mutation.…”

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confidence: 99%

Chance and necessity in the evolution of RNase P

Gopalan

Jarrous

Krasilnikov

2017

RNA

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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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Protein-only RNase P function in Escherichia coli: viability, processing defects and differences between PRORP isoenzymes

Cited by 21 publications

References 56 publications

Minimal and RNA-free RNase P in Aquifex aeolicus

Minimal and RNA-free RNase P in Aquifex aeolicus

Homologs of aquifex aeolicus protein‐only RNase P are not the major RNase P activities in the archaea haloferax volcanii and methanosarcina mazei

Chance and necessity in the evolution of RNase P

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