抄録
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.
| 元の言語 | 英語 |
|---|---|
| ページ（範囲） | 594-598 |
| ページ数 | 5 |
| ジャーナル | Biochemical and Biophysical Research Communications |
| 巻 | 440 |
| 発行部数 | 4 |
| DOI | |
| 出版物ステータス | 出版済み - 11 1 2013 |
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All Science Journal Classification (ASJC) codes
- Biophysics
- Biochemistry
- Molecular Biology
- Cell Biology
これを引用
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. / Hazeyama, Kohsuke; Ishihara, Masato; Ueda, Toshifumi; Nishimoto, Etsuko; Nakashima, Takashi; Kakuta, Yoshimitsu; Kimura, Makoto.
：: Biochemical and Biophysical Research Communications, 巻 440, 番号 4, 01.11.2013, p. 594-598.研究成果: ジャーナルへの寄稿 › 記事
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TY - JOUR
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
DO - 10.1016/j.bbrc.2013.09.140
M3 - Article
VL - 440
SP - 594
EP - 598
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 4
ER -