Substrate sequence effects on "hammerhead" RNA catalytic efficiency.

Abstract

The "hammerhead" RNA self-cleaving domain can be assembled from two RNA molecules: a large (.apprxeq. 34 nucleotide) ribozyme RNA containing most of the catalytically essential nucleotides and a small (.apprxeq. 13 nucleotide) substrate RNA containing the cleavage site. Four such hammerheads that contained identical catalytic core sequences but differed in the base composition of the helices that are involved in substrate binding had been reported to vary in cleavage rates by more than 70-fold under similar reaction conditions. Steady-state kinetic analyses reveal that kcat values are nearly the same for these hammerheads but Km values vary nearly 60-fold. The substrates for reaction having high Km values from aggregated that are virtually nonreactive. These observations demonstrate that the secondary structure of substrate RNA can be a major determinant of hammerhead catalytic efficiency.