Biochemical and Biophysical Research Communications, Vol.440, No.4, 594-598, 2013
Extra-structural elements in the RNA recognition motif in archaeal Pop5 play a crucial role in the activation of RNase P RNA from Pyrococcus horikoshii OT3
Ribonuclease P (RNase P) is a ribonucleoprotein complex essential for the processing of 5' leader sequences of precursor tRNAs (pre-tRNA). PhoPop5 is an archaeal homolog of human RNase P protein hPop5 involved in the activation of RNase P RNA (PhopRNA) in the hyperthermophilic archaeon Pyrococcus horikoshii, probably by promoting RNA annealing (AN) and RNA strand displacement (SD). Although PhoPop5 folds into the RNA recognition motif (RRM), it is distinct from the typical RRM in that it has an insertion of alpha-helix (alpha 2) between alpha 1 and beta 2. Biochemical and structural data have shown that the dimerization of PhoPop5 through the loop between alpha 1 and alpha 2 is required for the activation of PhopRNA. In addition, PhoPop5 has additional helices (alpha 4 and alpha 5) at the C-terminus, which pack against one face of the beta-sheet. In this study, we examined the contribution of the C-terminal helices to the activation of PhopRNA using mutation analyses. Reconstitution experiments and fluorescence resonance energy transfer (FRET)-based assays indicated that deletion of the C-terminal helices alpha 4 and alpha 5 significantly influenced on the pre-tRNA cleavage activity and abolished AN and SD activities, while that of alpha 5 had little effect on these activities. Moreover, the FRET assay showed that deletion of the loop between alpha 1 and alpha 2 had no influence on the AN and SD activity. Further mutational analyses suggested that basic residues at alpha 4 are involved in interaction with PhopRNA, while hydrophobic residues at alpha 4 participate in interaction with hydrophobic residues at the beta-sheet, thereby stabilizing an appropriate orientation of the helix alpha 4. Together, these results indicate that extra-structural elements in the RRM in PhoPop5 play a crucial role in the activation of PhopRNA. (C) 2013 Elsevier Inc. All rights reserved.