Effects of phosphorothioate modifications on precursor tRNA processing by eukaryotic RNase P enzymes.
The cleavage mechanism has been studied for nuclear RNase P from Saccharomyces cerevisiae, Homo sapiens sapiens and Dictyostelium discoideum, representing distantly related branches of the Eukarya. This was accomplished by using precursor tRNAs (ptRNAs) carrying a single Rp or Sp-phosphorothioate modification at the normal RNase P cleavage site (position -1/+1). All three eukaryotic RNase P enzymes cleaved the Sp-diastereomeric ptRNA exclusively one nucleotide upstream (position -2/-1) of the modified canonical cleavage site. Rp-diastereomeric ptRNA was cleaved with low efficiency at the modified -1/+1 site by human RNase P, at both the -2/-1 and -1/+1 site by yeast RNase P, and exclusively at the -2/-1 site by D. discoideum RNase P. The presence of Mn(2+ )and particularly Cd(2+) inhibited the activity of all three enzymes. Nevertheless, a Mn(2+ )rescue of cleavage at the modified -1/+1 site was observed with yeast RNase P and the Rp-diastereomeric ptRNA, consistent with direct metal ion coordination to the (pro)-Rp substituent during catalysis as observed for bacterial RNase P enzymes. In summary, our results have revealed common active-site constraints for eukaryotic and bacterial RNase P enzymes. In all cases, an Rp as well as an Sp-phosphorothioate modification at the RNase P cleavage site strongly interfered with the catalytic process, whereas substantial functional interference is essentially restricted to one of the two diastereomers in other RNA and protein-catalyzed hydrolysis reactions, such as those catalyzed by the Tetrahymena ribozyme and nuclease P1.
Pfeiffer
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Institut für Biochemie, Medizinische Universität zu Lübeck, Ratzeburger Allee 160, Lübeck, D-23538, Germany.
Tekos
A
A
Warnecke
J M
JM
Drainas
D
D
Engelke
D R
DR
Séraphin
B
B
Hartmann
R K
RK
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002104:Cadmium / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D002467:Cell Nucleus / Q000201:enzymology / Q000235:genetics; D004023:Dictyostelium / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D004722:Endoribonucleases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D005818:Genetic Engineering; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008956:Models, Chemical; D063086:Organothiophosphates / Q000378:metabolism*; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D043262:Ribonuclease P; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D063086:Organothiophosphates; D012322:RNA Precursors; D016337:RNA, Catalytic; D002104:Cadmium; D008345:Manganese; D012343:RNA, Transfer; D004722:Endoribonucleases; C468368:RPP14 protein, human; D043262:Ribonuclease P; D008274:Magnesium