Structure and mechanistic features of the prokaryotic minimal RNase P

  1. Rebecca Feyh
  2. Nadine Bianca Waeber
  3. Simone Prinz
  4. Pietro Ivan Giammarinaro
  5. Gert Bange
  6. Georg Hochberg
  7. Roland Karl Hartmann  Is a corresponding author
  8. Florian Altegoer  Is a corresponding author
  1. Institute of Pharmaceutical Chemistry, Philipps-University Marburg,, Germany
  2. Department of Structural Biology, Max Planck Institute of Biophysics, Germany
  3. Center for Synthetic Microbiology and Department of Chemistry, Philipps-University Marburg, Germany
  4. Max Planck Institute of Biophysics, Germany
  5. Philipps-Universitaet Marburg, Germany

Abstract

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.

Data availability

Coordinates and structure factors have been deposited within the protein data bank (PDB) and the electron microscopy data bank (EMDB) under accession codes: 7OG5 and EMD-12878. The authors declare that all other data supporting the findings of this study are available within the article and its supplementary information files.

Article and author information

Author details

  1. Rebecca Feyh

    Institute of Pharmaceutical Chemistry, Philipps-University Marburg,, Marburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Nadine Bianca Waeber

    Institute of Pharmaceutical Chemistry, Philipps-University Marburg,, Marburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Simone Prinz

    Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Pietro Ivan Giammarinaro

    Chemistry, Center for Synthetic Microbiology and Department of Chemistry, Philipps-University Marburg, Marburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0356-8481
  5. Gert Bange

    Max Planck Institute of Biophysics, Marburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Georg Hochberg

    Max Planck Institute of Biophysics, Marburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Roland Karl Hartmann

    Philipps-Universitaet Marburg, Marburg, Germany
    For correspondence
    roland.hartmann@staff.uni-marburg.de
    Competing interests
    The authors declare that no competing interests exist.
  8. Florian Altegoer

    Chemistry, Center for Synthetic Microbiology and Department of Chemistry, Philipps-University Marburg, Marburg, Germany
    For correspondence
    altegoer@uni-marburg.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6012-9047

Funding

Deutsche Forschungsgemeinschaft (HA 1672/19-1)

  • Roland Karl Hartmann

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Philip A Cole, Harvard Medical School, United States

Publication history

  1. Received: May 7, 2021
  2. Accepted: June 25, 2021
  3. Accepted Manuscript published: June 28, 2021 (version 1)
  4. Version of Record published: July 8, 2021 (version 2)

Copyright

© 2021, Feyh et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,387
    Page views
  • 225
    Downloads
  • 4
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Rebecca Feyh
  2. Nadine Bianca Waeber
  3. Simone Prinz
  4. Pietro Ivan Giammarinaro
  5. Gert Bange
  6. Georg Hochberg
  7. Roland Karl Hartmann
  8. Florian Altegoer
(2021)
Structure and mechanistic features of the prokaryotic minimal RNase P
eLife 10:e70160.
https://doi.org/10.7554/eLife.70160

Further reading

    1. Biochemistry and Chemical Biology
    Layla Drwesh et al.
    Research Article Updated

    Signal-anchored (SA) proteins are anchored into the mitochondrial outer membrane (OM) via a single transmembrane segment at their N-terminus while the bulk of the proteins is facing the cytosol. These proteins are encoded by nuclear DNA, translated on cytosolic ribosomes, and are then targeted to the organelle and inserted into its OM by import factors. Recently, research on the insertion mechanisms of these proteins into the mitochondrial OM have gained a lot of attention. In contrast, the early cytosolic steps of their biogenesis are unresolved. Using various proteins from this category and a broad set of in vivo, in organello, and in vitro assays, we reconstituted the early steps of their biogenesis. We identified a subset of molecular (co)chaperones that interact with newly synthesized SA proteins, namely, Hsp70 and Hsp90 chaperones and co-chaperones from the Hsp40 family like Ydj1 and Sis1. These interactions were mediated by the hydrophobic transmembrane segments of the SA proteins. We further demonstrate that interfering with these interactions inhibits the biogenesis of SA proteins to a various extent. Finally, we could demonstrate direct interaction of peptides corresponding to the transmembrane segments of SA proteins with the (co)chaperones and reconstitute in vitro the transfer of such peptides from the Hsp70 chaperone to the mitochondrial Tom70 receptor. Collectively, this study unravels an array of cytosolic chaperones and mitochondrial import factors that facilitates the targeting and membrane integration of mitochondrial SA proteins.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Rajesh Sharma et al.
    Research Article

    Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cGMP signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory pseudo-substrate sequences to PKG Iα and Iβ that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here we present a crystal structure of PKG Iβ in which the auto-inhibitory sequence and the cyclic nucleotide binding domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iβ auto-inhibitory sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I cyclic nucleotide binding domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wild type cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iβ auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs.