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RNase P RNA from the recently evolved plastid of Paulinella and from algae.

Bernal-Bayard P, Puerto-Galán L, Vioque A - Int J Mol Sci (2014)

Bottom Line: The RNase P RNA catalytic subunit (RPR) encoded in some plastids has been found to be functionally defective.The chromatophore RPR sequence deviates from consensus at some positions but those changes allow optimal activity compared with mutated chromatophore RPR with the consensus sequence.We have analyzed additional RPR sequences identifiable in plastids and have found that it is present in all red algae and in several prasinophyte green algae.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla and CSIC, Américo Vespucio 49, 41092 Sevilla, Spain. pilar.bernal@ibvf.csic.es.

ABSTRACT
The RNase P RNA catalytic subunit (RPR) encoded in some plastids has been found to be functionally defective. The amoeba Paulinella chromatophora contains an organelle (chromatophore) that is derived from the recent endosymbiotic acquisition of a cyanobacterium, and therefore represents a model of the early steps in the acquisition of plastids. In contrast with plastid RPRs the chromatophore RPR retains functionality similar to the cyanobacterial enzyme. The chromatophore RPR sequence deviates from consensus at some positions but those changes allow optimal activity compared with mutated chromatophore RPR with the consensus sequence. We have analyzed additional RPR sequences identifiable in plastids and have found that it is present in all red algae and in several prasinophyte green algae. We have assayed in vitro a subset of the plastid RPRs not previously analyzed and confirm that these organelle RPRs lack RNase P activity in vitro.

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Mutagenesis of RPR. (A) Highlight of helices P4 and P5 in RPR from P. chromatophora M0880 indicating the nucleotides that were changed to generate the different mutant RPRs and (B) Crystal structure of the same RNA region from Thermotoga maritima RNase P-tRNA complex (PDB ID:3Q1Q) [26]. Part of the tRNA substrate acceptor stem is shown (cyan). Nucleotides 1 of the tRNA (G1, cyan) as well as the three nucleotides that were mutated are depicted as spheres with the same colors as in (A) and in Figure 1. In T. maritima RPR there is an A-U base pair at the end of P4, and position equivalent to the chromatophore U237 is the consensus C. The two magnesium ions at the active center are depicted as yellow spheres. The structure was rendered with Pymol.
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ijms-15-20859-f004: Mutagenesis of RPR. (A) Highlight of helices P4 and P5 in RPR from P. chromatophora M0880 indicating the nucleotides that were changed to generate the different mutant RPRs and (B) Crystal structure of the same RNA region from Thermotoga maritima RNase P-tRNA complex (PDB ID:3Q1Q) [26]. Part of the tRNA substrate acceptor stem is shown (cyan). Nucleotides 1 of the tRNA (G1, cyan) as well as the three nucleotides that were mutated are depicted as spheres with the same colors as in (A) and in Figure 1. In T. maritima RPR there is an A-U base pair at the end of P4, and position equivalent to the chromatophore U237 is the consensus C. The two magnesium ions at the active center are depicted as yellow spheres. The structure was rendered with Pymol.

Mentions: As mentioned earlier, the chromatophore RPR has a C-U pair at the end of the highly conserved P4 helix (positions 55 and 370). What is more, nucleotide 237 is a U, instead of the highly conserved C at this position (Figure 4A). According to the crystal structure of bacterial RPR bound to tRNA (Figure 4B) [26], nucleotide 237 stacks on base pair 55-370, and they are very close to the active site, next to the first nucleotide of the tRNA and the two magnesium ions involved in catalysis. In order to assess the relevance of these otherwise conserved three nucleotides we have prepared several sequence variants of chromatophore RPR by site-directed mutagenesis and compared their RNase P activity in vitro under single turnover conditions with two different concentrations of RNA (0.05 and 4 µM) (Table 1).


RNase P RNA from the recently evolved plastid of Paulinella and from algae.

Bernal-Bayard P, Puerto-Galán L, Vioque A - Int J Mol Sci (2014)

Mutagenesis of RPR. (A) Highlight of helices P4 and P5 in RPR from P. chromatophora M0880 indicating the nucleotides that were changed to generate the different mutant RPRs and (B) Crystal structure of the same RNA region from Thermotoga maritima RNase P-tRNA complex (PDB ID:3Q1Q) [26]. Part of the tRNA substrate acceptor stem is shown (cyan). Nucleotides 1 of the tRNA (G1, cyan) as well as the three nucleotides that were mutated are depicted as spheres with the same colors as in (A) and in Figure 1. In T. maritima RPR there is an A-U base pair at the end of P4, and position equivalent to the chromatophore U237 is the consensus C. The two magnesium ions at the active center are depicted as yellow spheres. The structure was rendered with Pymol.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264200&req=5

ijms-15-20859-f004: Mutagenesis of RPR. (A) Highlight of helices P4 and P5 in RPR from P. chromatophora M0880 indicating the nucleotides that were changed to generate the different mutant RPRs and (B) Crystal structure of the same RNA region from Thermotoga maritima RNase P-tRNA complex (PDB ID:3Q1Q) [26]. Part of the tRNA substrate acceptor stem is shown (cyan). Nucleotides 1 of the tRNA (G1, cyan) as well as the three nucleotides that were mutated are depicted as spheres with the same colors as in (A) and in Figure 1. In T. maritima RPR there is an A-U base pair at the end of P4, and position equivalent to the chromatophore U237 is the consensus C. The two magnesium ions at the active center are depicted as yellow spheres. The structure was rendered with Pymol.
Mentions: As mentioned earlier, the chromatophore RPR has a C-U pair at the end of the highly conserved P4 helix (positions 55 and 370). What is more, nucleotide 237 is a U, instead of the highly conserved C at this position (Figure 4A). According to the crystal structure of bacterial RPR bound to tRNA (Figure 4B) [26], nucleotide 237 stacks on base pair 55-370, and they are very close to the active site, next to the first nucleotide of the tRNA and the two magnesium ions involved in catalysis. In order to assess the relevance of these otherwise conserved three nucleotides we have prepared several sequence variants of chromatophore RPR by site-directed mutagenesis and compared their RNase P activity in vitro under single turnover conditions with two different concentrations of RNA (0.05 and 4 µM) (Table 1).

Bottom Line: The RNase P RNA catalytic subunit (RPR) encoded in some plastids has been found to be functionally defective.The chromatophore RPR sequence deviates from consensus at some positions but those changes allow optimal activity compared with mutated chromatophore RPR with the consensus sequence.We have analyzed additional RPR sequences identifiable in plastids and have found that it is present in all red algae and in several prasinophyte green algae.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla and CSIC, Américo Vespucio 49, 41092 Sevilla, Spain. pilar.bernal@ibvf.csic.es.

ABSTRACT
The RNase P RNA catalytic subunit (RPR) encoded in some plastids has been found to be functionally defective. The amoeba Paulinella chromatophora contains an organelle (chromatophore) that is derived from the recent endosymbiotic acquisition of a cyanobacterium, and therefore represents a model of the early steps in the acquisition of plastids. In contrast with plastid RPRs the chromatophore RPR retains functionality similar to the cyanobacterial enzyme. The chromatophore RPR sequence deviates from consensus at some positions but those changes allow optimal activity compared with mutated chromatophore RPR with the consensus sequence. We have analyzed additional RPR sequences identifiable in plastids and have found that it is present in all red algae and in several prasinophyte green algae. We have assayed in vitro a subset of the plastid RPRs not previously analyzed and confirm that these organelle RPRs lack RNase P activity in vitro.

Show MeSH