Reconstitution of archaeal ribonuclease P from RNA and four protein components

Crystal Structure and Functional Analysis of an Archaeal Chromatin Protein Alba from the Hyperthermophilic ArchaeonPyrococcus horikoshiiOT3

“…Five RNase P proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38), PhopRNA, and 32 P-labeled pre-tRNA Tyr in P. horikoshii were prepared as described previously. 21,23) All nucleotide sequence data were cited in the P. horikoshii OT3 genome database (http://www.bio.nite.go.jp). All other chemicals were of analytical grade for biochemical use.…”

“…Cloning and expression of the resulting DNA fragment were done in a manner identical to those described for expression of P. horikoshii RNase P proteins. 21,23) E. coli lysate overproducing PhoAlba in 50 mM Tris-HCl (pH 7.0), containing 200 mM NaCl, was first heated at 80 C for 40 min, and then the supernatant was applied onto a SP-Sepharose FF column (1:5 Â 12 cm) equilibrated with 50 mM TrisHCl (pH 7.0), containing 200 mM NaCl. The protein was eluted with a linear gradient from 0.2 to 1.2 M NaCl in the starting buffer, and the purified protein was dialyzed against 50 mM Tris-HCl (pH 7.0) buffer, containing 200 mM NaCl, 10 mM MgCl 2 , and 200 mM arginine hydrochloride.…”

“…The RNA-binding capacity of PhoAlba for PhopRNA and pre-tRNA Tyr was tested by gel shift assay, as described previously. 21,23) The involvement of PhoAlba in pre-tRNA processing activity was analyzed as follows: reconstituted particles R-5P and R-4P, composed of PhopRNA and four and five proteins respectively, were prepared, and then an equimolar amount of PhoAlba was added. The pretRNA processing activity for R-5P and R-4P was analyzed at 55 C and 70 C respectively for the indicated periods of time using in vitro transcribed 32 P-labelled P. horikoshii pre-tRNA Tyr , as described previously.…”

“…The pretRNA processing activity for R-5P and R-4P was analyzed at 55 C and 70 C respectively for the indicated periods of time using in vitro transcribed 32 P-labelled P. horikoshii pre-tRNA Tyr , as described previously. 21,23) The optimum temperature of the reconstituted R-4P and PhoAlba was analyzed as described above, except that the activity was measured at various temperatures. Reaction products were separated on 15% polyacrylamide denaturing gels, and then visualized using a PhosphorImager, FLA-5000 (Fuji Film, Tokyo).…”

“…20) We have found via a reconstitution experiment that RNase P RNA (PhopRNA) and four proteins, PhoPop5, PhoRpp21, PhoRpp29, and PhoRpp30, are essential to the RNase P activity of the Pyrococcus horikoshii OT3. 21) The RNase P proteins from P. horikoshii OT3 were designated according to their homology to the corresponding human proteins, and the prefix Pho is added to differentiate them from homologous proteins from other organisms. 22) The reconstituted particle (R-4P), however, had a lower optimal temperature (around 55 C) than that (70 C) for the authentic RNase P from P. horikoshii.…”