In vitro selection for altered divalent metal specificity in the RNase P RNA.
The ribozyme RNase P absolutely requires divalent metal ions for catalytic function. Multiple Mg2+ ions contribute to the optimal catalytic efficiency of RNase P, and it is likely that the tertiary structure of the ribozyme forms a specific metal-binding pocket for these ions within the active-site. To identify base moieties that contribute to catalytic metal-binding sites, we have used in vitro selection to isolate variants of the Escherichia coli RNase P RNA with altered specificities for divalent metal. RNase P RNA variants with increased activity in Ca2+ were enriched over 18 generations of selection for catalysis in the presence of Ca2+, which is normally disfavored relative to Mg2+. Although a wide spectrum of mutations was found in the generation-18 clones, only a single point mutation was common to all clones: a cytosine-to-uracil transition at position 70 (E. coli numbering) of RNase P. Analysis of the C70U point mutant in a wild-type background confirmed that the identity of the base at position 70 is the sole determinant of Ca2+ selectivity. It is noteworthy that C70 lies within the phylogenetically well conserved J3/4-P4-J2/4 region, previously implicated in Mg2+ binding. Our finding that a single base change is sufficient to alter the metal preference of RNase P is further evidence that the J3/4-P4-J2/4 domain forms a portion of the ribozyme's active site.
Frank
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Department of Plant and Microbial Biology, 111 Koshland Hall, University of California at Berkeley, Berkeley, CA 94720-3102, USA.
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D001483:Base Sequence; D001665:Binding Sites; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D043262:Ribonuclease P; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D008670:Metals; D016337:RNA, Catalytic; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli