Functional characterization of archaeal homologs of human nuclear RNase P proteins Rpp21 and Rpp29 provides insights into the molecular basis of their cooperativity in catalysis

Dan Jiang, Kenta Izumi, Toshifumi Ueda, Kosuke Oshima, Takashi Nakashima, Makoto Kimura

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Ribonuclease P (RNase P) is a ribonucleoprotein that catalyzes the processing of 5′ leader sequences of precursor tRNAs (pre-tRNA). RNase P proteins PhoRpp21 and PhoRpp29 in the hyperthermophilic archaeon Pyrococcus horikoshii, homologs of human nuclear RNase P proteins Rpp21 and Rpp29 respectively, fold into a heterodimeric structure and synergistically function in the activation of the specificity domain (S-domain) in RNase P RNA (PhopRNA). To elucidate the molecular basis for their cooperativity, we first analyzed binding ability to PhopRNA using a pull-down assay. The result showed that PhoRpp21 is able to bind to PhopRNA in the absence of PhoRpp29, whereas PhoRpp29 alone has reduced affinity to PhopRNA, suggesting that PhoRpp21 primarily functions as a binding element for PhopRNA in the PhoRpp21-PhoRpp29 complex. Mutational analyses suggested that although individual positively charged clusters contribute little to the PhopRNA binding, Lys53, Lys54, and Lys56 at the N-terminal helix (α2) in PhoRpp21 and 10 C-terminal residues in PhoRpp29 are essential for PhopRNA activation. Moreover, deletion of a single stranded loop linking P11 and P12 helices in the PhopRNA S-domain impaired the PhoRpp21-PhoRpp29 complex binding to PhopRNA. Collectively, the present results suggest that PhoRpp21 binds the loop between P11 and P12 helices through overall positively charged clusters on the surface of the complex and serves as a scaffold for PhoRpp29 to optimize structural conformation of its N-terminal helix (α2) in PhoRpp21, as well as C-terminal residues in PhoRpp29, for RNase P activity.

Original languageEnglish
Pages (from-to)68-74
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume482
Issue number1
DOIs
Publication statusPublished - Jan 1 2017

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Ribonuclease P
Catalysis
Proteins
Pyrococcus horikoshii
Chemical activation
Ribonucleoproteins
Archaea
RNA Precursors
Scaffolds
Conformations
Assays
RNA
Processing

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Functional characterization of archaeal homologs of human nuclear RNase P proteins Rpp21 and Rpp29 provides insights into the molecular basis of their cooperativity in catalysis. / Jiang, Dan; Izumi, Kenta; Ueda, Toshifumi; Oshima, Kosuke; Nakashima, Takashi; Kimura, Makoto.

In: Biochemical and Biophysical Research Communications, Vol. 482, No. 1, 01.01.2017, p. 68-74.

Research output: Contribution to journalArticle

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abstract = "Ribonuclease P (RNase P) is a ribonucleoprotein that catalyzes the processing of 5′ leader sequences of precursor tRNAs (pre-tRNA). RNase P proteins PhoRpp21 and PhoRpp29 in the hyperthermophilic archaeon Pyrococcus horikoshii, homologs of human nuclear RNase P proteins Rpp21 and Rpp29 respectively, fold into a heterodimeric structure and synergistically function in the activation of the specificity domain (S-domain) in RNase P RNA (PhopRNA). To elucidate the molecular basis for their cooperativity, we first analyzed binding ability to PhopRNA using a pull-down assay. The result showed that PhoRpp21 is able to bind to PhopRNA in the absence of PhoRpp29, whereas PhoRpp29 alone has reduced affinity to PhopRNA, suggesting that PhoRpp21 primarily functions as a binding element for PhopRNA in the PhoRpp21-PhoRpp29 complex. Mutational analyses suggested that although individual positively charged clusters contribute little to the PhopRNA binding, Lys53, Lys54, and Lys56 at the N-terminal helix (α2) in PhoRpp21 and 10 C-terminal residues in PhoRpp29 are essential for PhopRNA activation. Moreover, deletion of a single stranded loop linking P11 and P12 helices in the PhopRNA S-domain impaired the PhoRpp21-PhoRpp29 complex binding to PhopRNA. Collectively, the present results suggest that PhoRpp21 binds the loop between P11 and P12 helices through overall positively charged clusters on the surface of the complex and serves as a scaffold for PhoRpp29 to optimize structural conformation of its N-terminal helix (α2) in PhoRpp21, as well as C-terminal residues in PhoRpp29, for RNase P activity.",
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AU - Jiang, Dan

AU - Izumi, Kenta

AU - Ueda, Toshifumi

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AU - Nakashima, Takashi

AU - Kimura, Makoto

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AB - Ribonuclease P (RNase P) is a ribonucleoprotein that catalyzes the processing of 5′ leader sequences of precursor tRNAs (pre-tRNA). RNase P proteins PhoRpp21 and PhoRpp29 in the hyperthermophilic archaeon Pyrococcus horikoshii, homologs of human nuclear RNase P proteins Rpp21 and Rpp29 respectively, fold into a heterodimeric structure and synergistically function in the activation of the specificity domain (S-domain) in RNase P RNA (PhopRNA). To elucidate the molecular basis for their cooperativity, we first analyzed binding ability to PhopRNA using a pull-down assay. The result showed that PhoRpp21 is able to bind to PhopRNA in the absence of PhoRpp29, whereas PhoRpp29 alone has reduced affinity to PhopRNA, suggesting that PhoRpp21 primarily functions as a binding element for PhopRNA in the PhoRpp21-PhoRpp29 complex. Mutational analyses suggested that although individual positively charged clusters contribute little to the PhopRNA binding, Lys53, Lys54, and Lys56 at the N-terminal helix (α2) in PhoRpp21 and 10 C-terminal residues in PhoRpp29 are essential for PhopRNA activation. Moreover, deletion of a single stranded loop linking P11 and P12 helices in the PhopRNA S-domain impaired the PhoRpp21-PhoRpp29 complex binding to PhopRNA. Collectively, the present results suggest that PhoRpp21 binds the loop between P11 and P12 helices through overall positively charged clusters on the surface of the complex and serves as a scaffold for PhoRpp29 to optimize structural conformation of its N-terminal helix (α2) in PhoRpp21, as well as C-terminal residues in PhoRpp29, for RNase P activity.

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