IN-VITRO SELECTION FOR ALTERED DIVALENT METAL SPECIFICITY IN THE RNASE-P RNA

The ribozyme RNase P absolutely requires divalent metal ions for catal ytic 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 wit hin the active-site. To identify base moieties that contribute to cata lytic metal-binding sites, we have used ill vitro selection to isolate variants of the Escherichia coli RNase P RNA with altered specificiti es 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+. A lthough a wide spectrum of mutations was found in the generation-IS cl ones, only a single paint mutation was common to all clones: a cytosin e-to-uracil transition at position 70 (E. coil 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 o f Ca2+ selectivity. It is noteworthy that C70 lies within the phylogen etically well conserved J3/4-P4-J2/4 region, previously implicated in Mg2+ binding. Our finding that a single base change is sufficient to a lter 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.