Structure and mechanistic features of the prokaryotic minimal RNase P

Endonucleolytic removal of 5'-leader sequences from tRNA precursor transcripts (pre-tRNAs) by RNase P is essential for protein synthesis. Beyond RNA-based RNase P enzymes, protein-only versions of the enzyme exert this function in various Eukarya (there termed PRORPs) and in some bacteria (Aquifex aeolicus and close relatives); both enzyme types belong to distinct subgroups of the PIN domain metallonuclease superfamily. Homologs of Aquifex RNase P (HARPs) are also expressed in some other bacteria and many archaea, where they coexist with RNA-based RNase P and do not represent the main RNase P activity. Here we solved the structure of the bacterial HARP from Halorhodospira halophila by cryo-EM revealing a novel screw-like dodecameric assembly. Biochemical experiments demonstrate that oligomerization is required for RNase P activity of HARPs. We propose that the tRNA substrate binds to an extended spike-helix (SH) domain that protrudes from the screw-like assembly to position the 5'-end in close proximity to the active site of the neighboring dimer. The structure suggests that eukaryotic PRORPs and prokaryotic HARPs recognize the same structural elements of pre-tRNAs (tRNA elbow region and cleavage site). Our analysis thus delivers the structural and mechanistic basis for pre-tRNA processing by the prokaryotic HARP system.

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Structure and mechanistic features of the prokaryotic minimal RNase P

10.1101/2021.05.07.443126 ◽

2021 ◽

Author(s):

Rebecca Feyh ◽

Nadine Bianca Waeber ◽

Simone Prinz ◽

Pietro Ivan Giammarinaro ◽

Gert Bange ◽

...

Keyword(s):

Rnase P ◽

Structural Elements ◽

Aquifex Aeolicus ◽

Trna Processing ◽

Close Proximity ◽

Mechanistic Basis ◽

Halorhodospira Halophila ◽

Close Relatives ◽

Trna Precursor ◽

Trna Substrate

Endonucleolytic removal of 5'-leader sequences from tRNA precursor transcripts (pre-tRNAs) by RNase P is essential for protein synthesis. Beyond RNA-based RNase P enzymes, protein-only versions of the enzyme exert this function in various Eukarya (there termed PRORPs) and in some bacteria (Aquifex aeolicus and close relatives); both enzyme types belong to distinct subgroups of the PIN domain metallonuclease superfamily. Homologs of Aquifex RNase P (HARPs) are also expressed in some other bacteria and many archaea, where they coexist with RNA-based RNase P and do not represent the main RNase P activity. Here we solved the structure of the bacterial HARP from Halorhodospira halophila by cryo-EM revealing a novel screw-like dodecameric assembly. Biochemical experiments demonstrate that oligomerization is required for RNase P activity of HARPs. We propose that the tRNA substrate binds to an extended spike-helix (SH) domain that protrudes from the screw-like assembly to position the 5'-end in close proximity to the active site of the neighboring dimer subunit. The structure suggests that eukaryotic PRORPs and prokaryotic HARPs recognize the same structural elements of pre-tRNAs (tRNA elbow region and cleavage site). Our analysis thus delivers the structural and mechanistic basis for pre-tRNA processing by the prokaryotic HARP system.

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Minimal and RNA-free RNase P in Aquifex aeolicus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1707862114 ◽

2017 ◽

Vol 114 (42) ◽

pp. 11121-11126 ◽

Cited By ~ 19

Author(s):

Astrid I. Nickel ◽

Nadine B. Wäber ◽

Markus Gößringer ◽

Marcus Lechner ◽

Uwe Linne ◽

...

Keyword(s):

Escherichia Coli ◽

Saccharomyces Cerevisiae ◽

Gene Transfer ◽

Horizontal Gene Transfer ◽

Rnase P ◽

Aquifex Aeolicus ◽

Trna Processing ◽

Hyperthermophilic Bacterium ◽

Domains Of Life

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.

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Faculty Opinions recommendation of RNase P without RNA: identification and functional reconstitution of the human mitochondrial tRNA processing enzyme.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1141875.598973 ◽

2009 ◽

Author(s):

Burckhard Seelig

Keyword(s):

Rnase P ◽

Mitochondrial Trna ◽

Trna Processing ◽

Processing Enzyme

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Synthetic Riboswitches for the Analysis of tRNA Processing by eukaryotic RNase P Enzymes

RNA ◽

10.1261/rna.078814.121 ◽

2022 ◽

pp. rna.078814.121

Author(s):

Anna Ender ◽

Nadine Grafl ◽

Tim Kolberg ◽

Sven Findeiss ◽

Peter F. Stadler ◽

...

Keyword(s):

Homo Sapiens ◽

Rnase P ◽

Single Stranded Region ◽

Domains Of Life ◽

The Individual ◽

The Impact ◽

Individual Enzyme ◽

Trna Precursor

Removal of the 5' leader region is an essential step in the maturation of tRNA molecules in all domains of life. This reaction is catalyzed by various RNase P activities, ranging from ribonucleoproteins with ribozyme activity to protein-only forms. In Escherichia coli, the efficiency of RNase P mediated cleavage can be controlled by computationally designed riboswitch elements in a ligand-dependent way, where the 5' leader sequence of a tRNA precursor is either sequestered in a hairpin structure or presented as a single-stranded region accessible for maturation. In the presented work, the regulatory potential of such artificial constructs is tested on different forms of eukaryotic RNase P enzymes – two protein-only RNase P enzymes (PRORP1 and PRORP2) from Arabidopsis thaliana and the ribonucleoprotein of Homo sapiens. The PRORP enzymes were analyzed in vitro as well as in vivo in a bacterial RNase P complementation system. We also tested in HEK293T cells whether the riboswitches remain functional with human nuclear RNase P. While the regulatory principle of the synthetic riboswitches applies for all tested RNase P enzymes, the results also show differences in the substrate requirements of the individual enzyme versions. Hence, such designed RNase P riboswitches represent a novel tool to investigate the impact of the structural composition of the 5'-leader on substrate recognition by different types of RNase P enzymes.

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