Journal: Elife / Year: 2021 Title: Structure and mechanistic features of the prokaryotic minimal RNase P. Authors: Rebecca Feyh / Nadine B Waeber / Simone Prinz / Pietro Ivan Giammarinaro / Gert Bange / Georg Hochberg / Roland K Hartmann / Florian Altegoer / 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- ...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 ( and close relatives); both enzyme types belong to distinct subgroups of the PIN domain metallonuclease superfamily. Homologs of 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 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.
H: RNA-free ribonuclease P J: RNA-free ribonuclease P F: RNA-free ribonuclease P B: RNA-free ribonuclease P D: RNA-free ribonuclease P L: RNA-free ribonuclease P E: RNA-free ribonuclease P C: RNA-free ribonuclease P G: RNA-free ribonuclease P K: RNA-free ribonuclease P I: RNA-free ribonuclease P A: RNA-free ribonuclease P
Evidence: assay for oligomerization, Mass photometry was performed to determine the dynamic oligomeric assembly
Type
Name
Symmetry operation
Number
identity operation
1_555
1
Buried area
34590 Å2
ΔGint
-170 kcal/mol
Surface area
73650 Å2
Method
PISA
-
Components
#1: Protein
RNA-freeribonucleaseP / RNA-free RNase P / Protein-only RNase P
Mass: 24051.338 Da / Num. of mol.: 12 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Halorhodospira halophila (strain DSM 244 / SL1) (bacteria) Strain: DSM 244 / SL1 / Gene: Hhal_2243 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A1WZ95, ribonuclease P
-
Experimental details
-
Experiment
Experiment
Method: ELECTRON MICROSCOPY
EM experiment
Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction
-
Sample preparation
Component
Name: Dodecameric assembly of minimal RNAseP system from Halorhodospira halophila Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weight
Value: 0.39 MDa / Experimental value: YES
Source (natural)
Organism: Halorhodospira halophila SL1 (bacteria)
Source (recombinant)
Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solution
pH: 8 Details: Solutions were prepared freshly and filtered through a 0.2 um filter
Buffer component
ID
Conc.
Name
Formula
Buffer-ID
1
100mM
potassiumchloride
KCl
1
2
20mM
Tris(hydroxymethyl)aminomethan
Tris
1
Specimen
Conc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was monodisperse
Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K / Details: Blot for 11s with blot force -1 before plunging
-
Electron microscopy imaging
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
Microscopy
Model: FEI TITAN KRIOS
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
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