Evidence for an RNA-based catalytic mechanism in eukaryotic nuclear ribonuclease P.
Ribonuclease P is the enzyme responsible for removing the 5'-leader segment of precursor transfer RNAs in all organisms. All eukaryotic nuclear RNase Ps are ribonucleoproteins in which multiple protein components and a single RNA species are required for activity in vitro as well as in vivo. It is not known, however, which subunits participate directly in phosphodiester-bond hydrolysis. The RNA subunit of nuclear RNase P is evolutionarily related to its catalytically active bacterial counterpart, prompting speculation that in eukaryotes the RNA may be the catalytic component. In the bacterial RNase P reaction, Mg(II) is required to coordinate the nonbridging phosphodiester oxygen(s) of the scissile bond. As a consequence, bacterial RNase P cannot cleave pre-tRNA in which the pro-Rp nonbridging oxygen of the scissile bond is replaced by sulfur. In contrast, the RNase P reaction in plant chloroplasts is catalyzed by a protein enzyme whose mechanism does not involve Mg(II) coordinated by the pro-Rp oxygen. To determine whether the mechanism of nuclear RNase P resembles more closely an RNA- or a protein-catalyzed reaction, we analyzed the ability of Saccharomyces cerevisiae nuclear RNase P to cleave pre-tRNA containing a sulfur substitution of the pro-Rp oxygen at the cleavage site. Sulfur substitution at this position prohibits correct cleavage of pre-tRNA. Cleavage by eukaryotic RNase P thus depends on the presence of a thio-sensitive ligand to the pro-Rp oxygen of the scissile bond, and is consistent with a common, RNA-based mechanism for the bacterial and eukaryal enzymes.
Thomas
B C
BC
Department of Molecular Biosciences, The University of Kansas, Lawrence 66045-2106, USA.
Chamberlain
J
J
Engelke
D R
DR
Gegenheimer
P
P
D001483:Base Sequence; D002384:Catalysis; D002467:Cell Nucleus / Q000201:enzymology*; D002712:Chlorides / Q000378:metabolism; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005057:Eukaryotic Cells / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D017895:Manganese Compounds / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D009946:Organothiophosphorus Compounds / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043262:Ribonuclease P; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D002712:Chlorides; D029968:Escherichia coli Proteins; D017895:Manganese Compounds; D009946:Organothiophosphorus Compounds; D012322:RNA Precursors; D016337:RNA, Catalytic; D012360:RNA, Transfer, Phe; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; C022543:ribonuclease T(2); D008274:Magnesium; C025340:manganese chloride; D010100:Oxygen