Dataset Information

Structure and mechanistic features of the prokaryotic minimal RNase P.

ABSTRACT: Endonucleolytic removal of 5'-leader sequences from tRNA precursor transcripts (pre-tRNAs) by ribonuclease P (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-electron microscopy, 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.

SUBMITTER: Feyh R

PROVIDER: S-EPMC8266387 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Structure and mechanistic features of the prokaryotic minimal RNase P.

Feyh Rebecca R Waeber Nadine B NB Prinz Simone S Giammarinaro Pietro Ivan PI Bange Gert G Hochberg Georg G Hartmann Roland K RK Altegoer Florian F

eLife 20210628

Endonucleolytic removal of 5'-leader sequences from tRNA precursor transcripts (pre-tRNAs) by ribonuclease P (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 (<i>Aquifex aeolicus</i> and close relatives); both enzyme types belong to distinct subgroups of the PIN domain metallonuclease superfamily. Homologs of <i>Aquifex</i> RNase P (HARPs) are also ...[more]

PMID: 34180399

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Project description:BackgroundCircadian clocks are found in organisms of almost all domains including photosynthetic Cyanobacteria, whereby large diversity exists within the protein components involved. In the model cyanobacterium Synechococcus elongatus PCC 7942 circadian rhythms are driven by a unique KaiABC protein clock, which is embedded in a network of input and output factors. Homologous proteins to the KaiABC clock have been observed in Bacteria and Archaea, where evidence for circadian behavior in these domains is accumulating. However, interaction and function of non-cyanobacterial Kai-proteins as well as homologous input and output components remain mainly unclear.ResultsUsing a universal BLAST analyses, we identified putative KaiC-based timing systems in organisms outside as well as variations within Cyanobacteria. A systematic analyses of publicly available microarray data elucidated interesting variations in circadian gene expression between different cyanobacterial strains, which might be correlated to the diversity of genome encoded clock components. Based on statistical analyses of co-occurrences of the clock components homologous to Synechococcus elongatus PCC 7942, we propose putative networks of reduced and fully functional clock systems. Further, we studied KaiC sequence conservation to determine functionally important regions of diverged KaiC homologs. Biochemical characterization of exemplary cyanobacterial KaiC proteins as well as homologs from two thermophilic Archaea demonstrated that kinase activity is always present. However, a KaiA-mediated phosphorylation is only detectable in KaiC1 orthologs.ConclusionOur analysis of 11,264 genomes clearly demonstrates that components of the Synechococcus elongatus PCC 7942 circadian clock are present in Bacteria and Archaea. However, all components are less abundant in other organisms than Cyanobacteria and KaiA, Pex, LdpA, and CdpA are only present in the latter. Thus, only reduced KaiBC-based or even simpler, solely KaiC-based timing systems might exist outside of the cyanobacterial phylum, which might be capable of driving diurnal oscillations.

Project description:The implication of reduced proteasomal function in neurodegenerative diseases combined with numerous studies showing the protective effects of increasing proteasome activity in animal models justify the need to understand how the proteasome is activated for protein degradation. The C-terminal HbYX motif is present on many proteasome binding proteins and functions to tether activators to the 20S core particle. Peptides with a HbYX motif can also autonomously activate 20S gate-opening to allow protein degradation, but the underlying allosteric molecular mechanism is not clear. We designed a HbYX-like dipeptide mimetic that represents only the fundamental components of the HbYX motif to allow rigorous elucidation of the underlying molecular mechanisms of HbYX induced 20S gate-opening in the archaeal and mamalian proteasome. By generating several high-resolution cryo-EM structures (e.g. 1.9Å) we identified multiple proteasome α subunit residues involved in HbYX-dependent activation and the conformational changes involved in gate-opening. In addition, we generated mutants probing these structural findings and identified specific point mutations that strongly activate the proteasome by partially mimicking a HbYX-bound state. These structures resolve 3 novel mechanistic features that are critical for allosteric α subunit conformational changes that ultimately trigger gate-opening: 1) rearrangement of the loop adjacent to K66, 2) inter- and intra- α subunit conformational changes and 3) a pair of IT residues on the α N-terminus in the 20S channel that alternate binding sites to stabilize the open and closed states. All gate-opening mechanisms appear to converge on this "IT switch". When stimulated by the mimetic, the human 20S can degrade unfolded proteins such as tau, and prevent proteasomal inhibition by toxic soluble oligomers. Collectively, the results presented here provide a mechanistic model of HbYX-dependent 20S gate-opening and offer proof of concept for the robust potential of HbYX-like small molecules to stimulate proteasome function, which could be useful to treat neurodegenerative diseases.

Dataset Information

Structure and mechanistic features of the prokaryotic minimal RNase P.

Publications

Structure and mechanistic features of the prokaryotic minimal RNase P.

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