Helix P4 is a divalent metal ion binding site in the conserved core of the ribonuclease P ribozyme.
The ribonuclease P ribozyme (RNase P RNA), like other large ribozymes, requires magnesium ions for folding and catalytic function; however, specific sites of metal ion coordination in RNase P RNA are not well defined. To identify and characterize individual nucleotide functional groups in the RNase P ribozyme that participate in catalytic function, we employed self-cleaving ribozyme-substrate conjugates that facilitate measurement of the effects of individual functional group modifications. The self-cleavage rates and pH dependence of two different ribozyme-substrate conjugates were determined and found to be similar to the single turnover kinetics of the native ribozyme. Using site-specific phosphorothioate substitutions, we provide evidence for metal ion coordination at the pro-Rp phosphate oxygen of A67, in the highly conserved helix P4, that was previously suggested by modification-interference experiments. In addition, we detect a new metal ion coordination site at the pro-Sp phosphate oxygen of A67. These findings, in combination with the proximity of A67 to the pre-tRNA cleavage site, support the conclusion that an important role of helix P4 in the RNase P ribozyme is to position divalent metal ions that are required for catalysis.
Christian
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Center for RNA Molecular Biology, Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Kaye
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Harris
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D001483:Base Sequence; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000378:metabolism*; D017124:Conserved Sequence / Q000235:genetics*; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D043262:Ribonuclease P; D013455:Sulfur / Q000378:metabolism; D013873:Thionucleotides / Q000378:metabolism
D002413:Cations, Divalent; D029968:Escherichia coli Proteins; D009843:Oligoribonucleotides; D010710:Phosphates; D012322:RNA Precursors; D016337:RNA, Catalytic; D013873:Thionucleotides; D002104:Cadmium; D008345:Manganese; D013455:Sulfur; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D008274:Magnesium; D010100:Oxygen