In vitro selection for altered divalent metal specificity in the RNase P RNA

摘要

The ribozyme RNase P absolutely requires divalent metal ions for catalytic function. Multiple Mg~2+ 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 Ca~2+ were enriched over 18 generations of selection for catalysis in the presence of Ca~2+, which is normally disfavored relative to Mg~2+.