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

Kohsuke Hazeyama, Masato Ishihara, Toshifumi Ueda, Etsuko Nishimoto, Takashi Nakashima, Yoshimitsu Kakuta, Makoto Kimura

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Abstract

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 α-helix (α2) between α1 and β2. Biochemical and structural data have shown that the dimerization of PhoPop5 through the loop between α1 and α2 is required for the activation of PhopRNA. In addition, PhoPop5 has additional helices (α4 and α5) at the C-terminus, which pack against one face of the β-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 α4 and α5 significantly influenced on the pre-tRNA cleavage activity and abolished AN and SD activities, while that of α5 had little effect on these activities. Moreover, the FRET assay showed that deletion of the loop between α1 and α2 had no influence on the AN and SD activity. Further mutational analyses suggested that basic residues at α4 are involved in interaction with PhopRNA, while hydrophobic residues at α4 participate in interaction with hydrophobic residues at the β-sheet, thereby stabilizing an appropriate orientation of the helix α4. Together, these results indicate that extra-structural elements in the RRM in PhoPop5 play a crucial role in the activation of PhopRNA.

Original languageEnglish
Pages (from-to)594-598
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume440
Issue number4
DOIs
Publication statusPublished - Nov 1 2013

Fingerprint

Pyrococcus horikoshii
Ribonuclease P
Fluorescence Resonance Energy Transfer
Chemical activation
RNA Precursors
RNA
Ribonucleoproteins
Archaea
Dimerization
Annealing
Hydrophobic and Hydrophilic Interactions
Assays
Mutation
RNA Recognition Motif
Processing

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "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",
abstract = "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 α-helix (α2) between α1 and β2. Biochemical and structural data have shown that the dimerization of PhoPop5 through the loop between α1 and α2 is required for the activation of PhopRNA. In addition, PhoPop5 has additional helices (α4 and α5) at the C-terminus, which pack against one face of the β-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 α4 and α5 significantly influenced on the pre-tRNA cleavage activity and abolished AN and SD activities, while that of α5 had little effect on these activities. Moreover, the FRET assay showed that deletion of the loop between α1 and α2 had no influence on the AN and SD activity. Further mutational analyses suggested that basic residues at α4 are involved in interaction with PhopRNA, while hydrophobic residues at α4 participate in interaction with hydrophobic residues at the β-sheet, thereby stabilizing an appropriate orientation of the helix α4. Together, these results indicate that extra-structural elements in the RRM in PhoPop5 play a crucial role in the activation of PhopRNA.",
author = "Kohsuke Hazeyama and Masato Ishihara and Toshifumi Ueda and Etsuko Nishimoto and Takashi Nakashima and Yoshimitsu Kakuta and Makoto Kimura",
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T1 - 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

AU - Hazeyama, Kohsuke

AU - Ishihara, Masato

AU - Ueda, Toshifumi

AU - Nishimoto, Etsuko

AU - Nakashima, Takashi

AU - Kakuta, Yoshimitsu

AU - Kimura, Makoto

PY - 2013/11/1

Y1 - 2013/11/1

N2 - 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 α-helix (α2) between α1 and β2. Biochemical and structural data have shown that the dimerization of PhoPop5 through the loop between α1 and α2 is required for the activation of PhopRNA. In addition, PhoPop5 has additional helices (α4 and α5) at the C-terminus, which pack against one face of the β-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 α4 and α5 significantly influenced on the pre-tRNA cleavage activity and abolished AN and SD activities, while that of α5 had little effect on these activities. Moreover, the FRET assay showed that deletion of the loop between α1 and α2 had no influence on the AN and SD activity. Further mutational analyses suggested that basic residues at α4 are involved in interaction with PhopRNA, while hydrophobic residues at α4 participate in interaction with hydrophobic residues at the β-sheet, thereby stabilizing an appropriate orientation of the helix α4. Together, these results indicate that extra-structural elements in the RRM in PhoPop5 play a crucial role in the activation of PhopRNA.

AB - 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 α-helix (α2) between α1 and β2. Biochemical and structural data have shown that the dimerization of PhoPop5 through the loop between α1 and α2 is required for the activation of PhopRNA. In addition, PhoPop5 has additional helices (α4 and α5) at the C-terminus, which pack against one face of the β-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 α4 and α5 significantly influenced on the pre-tRNA cleavage activity and abolished AN and SD activities, while that of α5 had little effect on these activities. Moreover, the FRET assay showed that deletion of the loop between α1 and α2 had no influence on the AN and SD activity. Further mutational analyses suggested that basic residues at α4 are involved in interaction with PhopRNA, while hydrophobic residues at α4 participate in interaction with hydrophobic residues at the β-sheet, thereby stabilizing an appropriate orientation of the helix α4. Together, these results indicate that extra-structural elements in the RRM in PhoPop5 play a crucial role in the activation of PhopRNA.

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U2 - 10.1016/j.bbrc.2013.09.140

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M3 - Article

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