Cloned (Comment) | Organism |
---|---|
gene pnp, cloned in a plasmid with rne gene, encoding endoribonuclease RNase E, fragments, expression of N-terminally GST- or His6-tagged enzyme fragments in Escherichia coli | Caulobacter vibrioides |
Crystallization (Comment) | Organism |
---|---|
purified recombinant RNA-free and RNA-bound PNPase, as protein-peptide and protein-RNA complexes, mixing of PNPase recognition peptide from RNase E, KPRRGWWRR, with apo-PNPase in a 2:1 ratio, sitting drop vapour diffusion method, mixing of equal volumes of protein solution and cyrstallization solution, containing w/v PEG 3350, 0.1 M Bis-Tris, pH 5.5, 0.1 M ammonium acetate for RNA-bound crystals, and with the apo-enzyme containing w/v PEG3000, 0.1 M trisodium citrate, pH 5.5, for hexagonal crystals and w/v PEG 3350, 0.15 M DL-malic acid for rhombohedral crystals, 18°C, 1 week, X-ray diffraction structure determination aand analysis at 2.6-3.3 A resolution, molecular replacement | Caulobacter vibrioides |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Caulobacter vibrioides | - |
gene pnp | - |
Caulobacter vibrioides NA1000 | - |
gene pnp | - |
Purification (Comment) | Organism |
---|---|
recombinant N-terminally GST-tagged enzyme fragments from Escherichia coli by glutathione affinity chromatography, of His6-tagged enzyme fragments by nickel affinity chromatography, followed by gel filtration in both procedures | Caulobacter vibrioides |
Subunits | Comment | Organism |
---|---|---|
trimer | the enzyme has a ring-like, trimeric architecture that creates a central channel where phosphorolytic active sites reside. One face of the ring is decorated with RNA-binding K-homology (KH) and S1 domains, domain organization with modular organization of conserved structural domains, overview | Caulobacter vibrioides |
Synonyms | Comment | Organism |
---|---|---|
PNPase | - |
Caulobacter vibrioides |
polynucleotide phosphorylase | - |
Caulobacter vibrioides |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Caulobacter vibrioides |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Caulobacter vibrioides |
General Information | Comment | Organism |
---|---|---|
evolution | two domains, both resembling closely the phosphorolytic exoribonuclease RNase PH, EC 27.7.56, almost certainly have originated from duplication and fusion of an ancestral gene. While the C-terminal RNase PH-like domain catalyses phosphorolytic attack of RNA, the N-terminal domain has lost this capacity. Instead, it contributes to the ring-like quaternary structure of the trimeric PNPase assembly | Caulobacter vibrioides |
additional information | the enzyme has a ring-like, trimeric architecture that creates a central channel where phosphorolytic active sites reside, with asymmetry within the catalytic core of the enzyme. One face of the ring is decorated with RNA-binding K-homology (KH) and S1 domains. In the RNA-free form, the S1 domains adopt a splayed conformation that may facilitate capture of RNA substrates. In the RNA-bound structure, the three KH domains collectively close upon the RNA and direct the 3' end towards a constricted aperture at the entrance of the central channel. Structural non-equivalence, induced upon RNA binding, helps to channel substrate to the active sites through mechanical ratcheting. Access to the PNPase active sites is through the central channel, which can accommodate single-stranded RNA with some structural adjustment of a constricted aperture at the channel entrance, residues and motifs involved in RNA directionality, recognition, and quarternary changes in the core, structure-function-relationship, detailed overview | Caulobacter vibrioides |
physiological function | polynucleotide phosphorylase is an exoribonuclease that cleaves single-stranded RNA substrates with 3' -5' directionality and processive behaviour | Caulobacter vibrioides |