A fifth protein subunit Ph1496p elevates the optimum temperature for the ribonuclease P activity from Pyrococcus horikoshii OT3.
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 degrees C), as compared with 70 degrees 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 degrees 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 A resolution by the molecular replacement method using ribosomal protein L7Ae from Haloarcula marismortui as a search model. Ph1496p comprises five alpha-helices and a four stranded beta-sheet. The beta-sheet is sandwiched by three helices (alpha1, alpha4, and alpha5) at one side and two helices (alpha2 and alpha3) 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.
Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan.
Hideo
H
Fukuhara
Mayumi
M
Kifusa
Mitsutoshi
M
Watanabe
Atsushi
A
Terada
Takashi
T
Honda
Tomoyuki
T
Numata
Yoshimitsu
Y
Kakuta
Makoto
M
Kimura
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D043902:Pyrococcus horikoshii / Q000201:enzymology* / Q000235:genetics; D043262:Ribonuclease P / Q000737:chemistry / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D013696:Temperature
D019843:Archaeal Proteins; D021122:Protein Subunits; D012269:Ribosomal Proteins; D043262:Ribonuclease P