Minimal and RNA-free RNase P in <em>Aquifex aeolicus</em>

Astrid Nickel

Nadine Wäber

Markus Gößringer

Marcus Lechner

Supplementary Material

Supplementary File

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^{Institute of Pharmaceutical Chemistry, Philipps-Universität Marburg, 35037 Marburg, Germany;}

^{Faculty of Chemistry, Mass Spectrometry, Philipps-Universität Marburg, 35032 Marburg, Germany;}

^{Center for Anatomy & Cell Biology, Medical University of Vienna, 1090 Vienna, Austria}

^{To whom correspondence should be addressed. Email:}ed.grubram-inu.ffats@nnamtrah.dnalor.

Edited by Sidney Altman, Yale University, New Haven, CT, and approved September 11, 2017 (received for review May 12, 2017)

Author contributions: A.I.N., N.B.W., M.G., W.R., and R.K.H. designed research; A.I.N., N.B.W., M.G. and U.T. performed research; U.L. contributed new reagents/analytic tools; A.I.N., N.B.W., M.G., M.L., W.R., and R.K.H. analyzed data; M.G., M.L., W.R., and R.K.H. wrote the paper.

Edited by Sidney Altman, Yale University, New Haven, CT, and approved September 11, 2017 (received for review May 12, 2017)

Significance

RNase P is a tRNA-processing enzyme of unique architectural diversity: either a catalytic RNA plus one or more (up to 10) proteins, or one (or three) unrelated proteins only. We identified yet another enzyme form in the bacterium Aquifex aeolicus, a 23-kDa protein and the smallest known form of RNase P. Apparently, it was acquired by horizontal gene transfer from Archaea. In some other bacteria and many archaea, it is simultaneously present with the presumably more ancient RNA-based enzyme form. Bacteria with both activities may represent the missing link of RNase P evolution, a transition state that had also been once traversed by the Aquificaceae, which, however, later lost their RNA-based RNase P.

Keywords: protein-only RNase P, Aquifex aeolicus, tRNA processing, HARP

Significance

Abstract

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.

Abstract

The 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.

RNP RNase P (RNA alone and holoenzyme) and HARP in vitro activities were tested as described in SI Appendix. —, enzyme not present; +(++), relative enzyme activities in processing assays or relative efficiencies in genetic complementation of strain BW; for example, in RNA-alone reactions, the k_obs of pretRNA^{cleavage was 0.54 min}^{(+++) for}E. coli and 0.11 min^{(+) for}T. indicus RNase P RNA; in complementation experiments (HARP in vivo), +++ was assigned to colony densities roughly corresponding to those obtained for BW bacteria complemented with aq_880 after overnight incubation at 37 °C.

Acknowledgments

We thank Diogo Monteiro for his help with the yeast complementation experiments and Dominik Helmecke for support with respect to processing assays, Johann Heider and Wolfgang Buckel for giving us access to the French press and ultracentrifuge, and Tina Krieg for technical assistance in sample preparation for mass spectrometry. The Superose 6 column was a loan from Peter Friedhoff, Justus-Liebig-Universität Gießen. A. aeolicus ribosomal subunits were kindly prepared in the laboratory of Ciarán Condon (Paris).

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1707862114/-/DCSupplemental.

Footnotes

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