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

Abstract

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

Structure and mechanistic features of the prokaryotic minimal RNase P

“…The active sites of both molecules superpose well with a root mean square deviation (r.m.s.d) of 0.486 over 30 Ca atoms. In earlier studies we could already show that the catalytic aspartates D7, D138, D142 and D161 are indispensable for Aq880 activity (Nickel et al 2017;Schwarz et al, 2019). Our Hhal2243 structure supports the prediction that these residues constitute the active site of the protein (Fig.…”

Structure and mechanistic features of the prokaryotic minimal RNase P

“…More precisely, the β-strands 5, 6, and 8, and the α-helices 16 and 20 within the metallonuclease domain of Arabidopsis thaliana PRORP1 ( At PRORP1) were used for the structural superposition with β1, β4, α1, and α6 of Hhal2243, as the overall scaffold of the two metallonuclease domains is related but shows large structural deviations. In earlier studies, we could already show that the catalytic aspartates D7, D138, D142, and D161 are indispensable for Aq880 activity ( Nickel et al, 2017 ; Schwarz et al, 2019 ). Our Hhal2243 structure supports the prediction that these residues constitute the active site of the protein ( Figure 3B ), including an almost perfect superposition with three of the four active site aspartates of At PRORP1 (D399, D475, and D493; Howard et al, 2012 ; Figure 3B ).…”

“…However, all of these HARP-positive archaea also encode the RNA and protein subunits of the RNA-based RNase P ( Nickel et al, 2017 ; Daniels et al, 2019 ). Remarkably, HARP gene knockouts in two Euryarchaeota, Haloferax volcanii and Methanosarcina mazei , showed no growth phenotypes under standard conditions, temperature, and salt stress ( H. volcanii ) or nitrogen deficiency ( M. mazei ) ( Schwarz et al, 2019 ). In contrast, it was impossible to entirely erase the RNase P RNA gene from the polyploid genome of H. volcanii (~18 genome copies per cell in exponential growth phase; Breuert et al, 2006 ).…”

“…The findings suggest that HARP is neither essential nor represents the housekeeping RNase P function in archaea, explaining its sporadic loss in archaea. HARPs are evolutionarily linked to toxin-antitoxin systems ( Daniels et al, 2019 ; Schwarz et al, 2019 ; Gobert et al, 2019 ). Frequently, the toxin proteins are endoribonucleases that cleave mRNA, rRNA, tmRNA, or tRNA to inhibit protein biosynthesis in response to certain stresses ( Masuda and Inouye, 2017 ).…”

Minimal protein-only RNase P structure reveals insights into tRNA precursor recognition and catalysis

“…According to the amino acid sequence of HARPs, they do not appear to contain a pre-tRNA-binding domain, such as the PPR domain of eukaryotic PRORPs. However, HARPs can catalyze the endonucleolytic maturation of pre-tRNA and substitute for RNP RNase P activity in Escherichia coli, as well as in Saccharomyces cerevisiae (11,19). How HARPs specifically recognize pre-tRNA molecules and cleave the 5 0 leader sequence of pre-tRNA is unknown.…”