A fifth protein subunit Ph1496p elevates the optimum temperature for the ribonuclease P activity from Pyrococcus horikoshii OT3

Hideo Fukuhara, Mayumi Kifusa, Mitsutoshi Watanabe, Atsushi Terada, Takashi Honda, Tomoyuki Numata, Yoshimitsu Kakuta, Makoto Kimura

Research output: Contribution to journalArticlepeer-review

54 Citations (Scopus)

Abstract

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5′ leader sequence of precursor tRNA. We previously found that the reconstituted particle (RP) composed of RNase P RNA and four proteins (Ph1481p, Ph1601p, Ph1771p, and Ph1877p) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 exhibited the RNase P activity, but had a lower optimal temperature (around at 55 °C), as compared with 70 °C of the authentic RNase P from P. horikoshii [Kouzuma et al., Biochem. Biophys. Res. Commun. 306 (2003) 666-673]. In the present study, we found that addition of a fifth protein Ph1496p, a putative ribosomal protein L7Ae, to RP specifically elevated the optimum temperature to about 70 °C comparable to that of the authentic RNase P. Characterization using gel shift assay and chemical probing localized Ph1496p binding sites on two stem-loop structures encompassing nucleotides A116-G201 and G229-C276 in P. horikoshii RNase P RNA. Moreover, the crystal structure of Ph1496p was determined at 2.0 Å resolution by the molecular replacement method using ribosomal protein L7Ae from Haloarcula marismortui as a search model. Ph1496p comprises five α-helices and a four stranded β-sheet. The β-sheet is sandwiched by three helices (α1, α4, and α5) at one side and two helices (α2 and α3) at other side. The archaeal ribosomal protein L7Ae is known to be a triple functional protein, serving as a protein component in ribosome and ribonucleoprotein complexes, box C/D, and box H/ACA. Although we have at present no direct evidence that Ph1496p is a real protein component in the P. horikoshii RNase P, the present result may assign an RNase P protein to L7Ae as a fourth function.

Original languageEnglish
Pages (from-to)956-964
Number of pages9
JournalBiochemical and Biophysical Research Communications
Volume343
Issue number3
DOIs
Publication statusPublished - May 12 2006

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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