PMID:9405616
| Citation |
Frank, DN and Pace, NR (1997) In vitro selection for altered divalent metal specificity in the RNase P RNA. Proc. Natl. Acad. Sci. U.S.A. 94:14355-60 |
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| Abstract |
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. |
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| Keywords |
Base Sequence; Binding Sites; Endoribonucleases/genetics; Endoribonucleases/metabolism; Enzyme Activation; Escherichia coli/enzymology; Escherichia coli Proteins; Metals/metabolism; Molecular Sequence Data; Mutation; RNA, Catalytic/genetics; RNA, Catalytic/metabolism; Ribonuclease P; Substrate Specificity |
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