Spinach chloroplast RNase P: a putative protein enzyme.
Ribonuclease P (RNase P) is the enzyme responsible for endonucleolytically separating the 5'-leader sequence from precursor tRNA molecules. In bacteria, and in the nuclei and mitochondria of all eukaryotes studied so far, RNase P contains an RNA subunit which is necessary for activity in vitro and in vivo. In contrast, we showed earlier that partially-purified RNase P from spinach chloroplasts had physical properties inconsistent with the presence of any RNA. We now report that the properties of the chloroplast enzyme, after 500 to 1500-fold purification, are consistent with enzymatic activity residing in a approximately 70 kDa polypeptide. Gel filtration chromatography on Sephacryl S-200 and S-300 provides a mass for chloroplast RNase P of approximately 70 +/- 5 kDa. A single polypeptide of approximately 70-80 kDa can be crosslinked to iodoUMP-substituted pre-tRNA. The labeling intensity of this polypeptide corresponds closely to the peak of RNase P activity on Sephacryl S-200 chromatography. Unlike the bacterial ribozyme-type RNase P, chloroplast RNase P is not a metalloenzyme. We showed previously that phosphodiester bond cleavage by the E. coli RNA enzyme absolutely requires Mg2+ or Mn2+ coordinated to the pro-Rp oxygen of the scissile phosphodiester phosphate. In contrast, we now find that chloroplast RNase P has no such requirement, and can accurately and efficiently cleave pre-tRNA containing an Rp-thio-substitution at the scissile bond. These data are entirely consistent with the hypothesis that RNase P in plant chloroplasts is not a ribozyme, but a conventional protein enzyme.
Thomas
B C
BC
Department of Biochemistry, University of Kansas, Lawrence 66045-2106, USA.
Gao
L
L
Stomp
D
D
Li
X
X
Gegenheimer
P A
PA
D005075:Biological Evolution; D002736:Chloroplasts / Q000201:enzymology*; D002850:Chromatography, Gel; D003429:Cross Reactions; D004722:Endoribonucleases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D000939:Epitopes; D006801:Humans; D008970:Molecular Weight; D010940:Plant Proteins / Q000276:immunology / Q000302:isolation & purification; D016337:RNA, Catalytic / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D043262:Ribonuclease P; D018724:Spinacia oleracea / Q000201:enzymology*
D000939:Epitopes; D010940:Plant Proteins; D016337:RNA, Catalytic; D004722:Endoribonucleases; C468368:RPP14 protein, human; D043262:Ribonuclease P