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 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.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Pyrococcus horikoshii/enzymology , Ribonuclease P/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Amino Acid Sequence , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Protein Subunits/chemistry , Protein Subunits/metabolism , Pyrococcus horikoshii/genetics , Ribonuclease P/chemistry , Sequence Alignment , TemperatureABSTRACT
Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5' leader sequence of precursor tRNA (pre-tRNA). RNase P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of RNA and five protein subunits (Ph1481p, Ph1496p, Ph1601p, Ph1771p, and Ph1877p). In vivo interactions among five protein subunits of RNase P in P. horikoshii OT3 were examined using a yeast two-hybrid system. The analysis indicates that proteins Ph1481p and Ph1601p interact strongly with Ph1877p and Ph1771p respectively, whereas Ph1481p interacts moderately with Ph1601p. In contrast, no interaction was detected between Ph1496p and the other four proteins. Co-immunoprecipitation analysis confirmed the interactions obtained by yeast two-hybrid assay.
