“…Figure 4.Surface representations of the RNase P with fitted subunits: ( A ) Panel 1: heterotetramer [2CZV (29)] of the archaeal homologs of Pop5 (dark blue) and Rpp1 (light blue) are fitted to the rear module. Panel 2: model of S. pombe RNA (60) (magenta) and archaeal homologs of the Pop4–Rpr2 dimer [2ZAE, green (27)] fitted to the base plate. Panel 3: Pop6 (orange), Pop7 (yellow) and P3 loop (purple) [3IAB, (35)] fitted to the foot domain.…”
Section: Resultsmentioning
confidence: 99%
“…Some components [Pop6–Pop7–P3 subcomplex (35) and the archaeal homolog of the 2:2 Pop5–Rpp1 subcomplex (29)] were fitted automatically to the segmented map using the Segger option of Chimera (fit to groups of regions). Other components [model of the Schizosaccharomyces pombe RNA (60), the archaeal homolog of the Rpr2–Pop4 subcomplex (27) and the L7Ae protein, which is homologous to Pop3 (34)] were placed manually, taking constrains from published interaction studies and our antibody labeling into account. The manual fits were further optimized with the fit to map option of Chimera, which performs a local optimization.…”
Section: Methodsmentioning
confidence: 99%
“…These data are complemented by protein–protein interaction studies in different species (37–41), which among other interactions repeatedly report interactions between homologs of Pop4–Pop5, Pop4–Rpr2, Pop5–Rpp1 and Pop6–Pop7. Apart from the Pop4–Pop5 subcomplex, all of the predicted binary complexes have been crystallized either from archaea or from yeast (27,29,35). …”
Ribonuclease P (RNase P) and RNase MRP are closely related ribonucleoprotein enzymes, which process RNA substrates including tRNA precursors for RNase P and 5.8 S rRNA precursors, as well as some mRNAs, for RNase MRP. The structures of RNase P and RNase MRP have not yet been solved, so it is unclear how the proteins contribute to the structure of the complexes and how substrate specificity is determined. Using electron microscopy and image processing we show that eukaryotic RNase P and RNase MRP have a modular architecture, where proteins stabilize the RNA fold and contribute to cavities, channels and chambers between the modules. Such features are located at strategic positions for substrate recognition by shape and coordination of the cleaved-off sequence. These are also the sites of greatest difference between RNase P and RNase MRP, highlighting the importance of the adaptation of this region to the different substrates.
“…Figure 4.Surface representations of the RNase P with fitted subunits: ( A ) Panel 1: heterotetramer [2CZV (29)] of the archaeal homologs of Pop5 (dark blue) and Rpp1 (light blue) are fitted to the rear module. Panel 2: model of S. pombe RNA (60) (magenta) and archaeal homologs of the Pop4–Rpr2 dimer [2ZAE, green (27)] fitted to the base plate. Panel 3: Pop6 (orange), Pop7 (yellow) and P3 loop (purple) [3IAB, (35)] fitted to the foot domain.…”
Section: Resultsmentioning
confidence: 99%
“…Some components [Pop6–Pop7–P3 subcomplex (35) and the archaeal homolog of the 2:2 Pop5–Rpp1 subcomplex (29)] were fitted automatically to the segmented map using the Segger option of Chimera (fit to groups of regions). Other components [model of the Schizosaccharomyces pombe RNA (60), the archaeal homolog of the Rpr2–Pop4 subcomplex (27) and the L7Ae protein, which is homologous to Pop3 (34)] were placed manually, taking constrains from published interaction studies and our antibody labeling into account. The manual fits were further optimized with the fit to map option of Chimera, which performs a local optimization.…”
Section: Methodsmentioning
confidence: 99%
“…These data are complemented by protein–protein interaction studies in different species (37–41), which among other interactions repeatedly report interactions between homologs of Pop4–Pop5, Pop4–Rpr2, Pop5–Rpp1 and Pop6–Pop7. Apart from the Pop4–Pop5 subcomplex, all of the predicted binary complexes have been crystallized either from archaea or from yeast (27,29,35). …”
Ribonuclease P (RNase P) and RNase MRP are closely related ribonucleoprotein enzymes, which process RNA substrates including tRNA precursors for RNase P and 5.8 S rRNA precursors, as well as some mRNAs, for RNase MRP. The structures of RNase P and RNase MRP have not yet been solved, so it is unclear how the proteins contribute to the structure of the complexes and how substrate specificity is determined. Using electron microscopy and image processing we show that eukaryotic RNase P and RNase MRP have a modular architecture, where proteins stabilize the RNA fold and contribute to cavities, channels and chambers between the modules. Such features are located at strategic positions for substrate recognition by shape and coordination of the cleaved-off sequence. These are also the sites of greatest difference between RNase P and RNase MRP, highlighting the importance of the adaptation of this region to the different substrates.
“…aPop4 + aRpr2 (P. horikoshii OT3, 2.2 Å , PDB ID 2ZAE [Honda et al 2008]; P. furiosus, NMR, PDB ID 2KI7 [Xu et al 2009]); and aPop5 + aRpp1 (P. horikoshii OT3, a heterotetramer, 2.0 Å , PDB ID 2CZV [Kawano et al 2006]). …”
Section: Proteins In Archaeal Rnase P Holoenzymesmentioning
confidence: 99%
“…5G; Honda et al 2008;Xu et al 2009) contains patches of phylogenetically conserved basic residues (Fig. 5H), which may constitute an RNA-binding surface (Xu et al 2009); however, details of RNA-protein interactions are not known.…”
Section: Folds Of Archaeal Rnase P Proteinsmentioning
Nuclear ribonuclease (RNase) P is a ubiquitous essential ribonucleoprotein complex, one of only two known RNA-based enzymes found in all three domains of life. The RNA component is the catalytic moiety of RNases P across all phylogenetic domains; it contains a well-conserved core, whereas peripheral structural elements are diverse. RNA components of eukaryotic RNases P tend to be less complex than their bacterial counterparts, a simplification that is accompanied by a dramatic reduction of their catalytic ability in the absence of protein. The size and complexity of the protein moieties increase dramatically from bacterial to archaeal to eukaryotic enzymes, apparently reflecting the delegation of some structural functions from RNA to proteins and, perhaps, in response to the increased complexity of the cellular environment in the more evolutionarily advanced organisms; the reasons for the increased dependence on proteins are not clear. We review current information on RNase P and the closely related universal eukaryotic enzyme RNase MRP, focusing on their functions and structural organization.
We demonstrate that surface‐induced dissociation (SID) coupled with ion mobility mass spectrometry (IM‐MS) is a powerful tool for determining the stoichiometry of a multi‐subunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg2+. We investigated Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5′ maturation. Previous step‐wise, Mg2+‐dependent reconstitutions of Pfu RNase P with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21⋅RPP29 and POP5⋅RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins showed RPP21⋅RPP29 and (POP5⋅RPP30)2 complexes, but indicated a 1:1 composition for all subunits when either one or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM‐MS in resolving conformational heterogeneity and yielding insights on RNP assembly.
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