BUILDING A KINETIC FRAMEWORK FOR GROUP-II INTRON RIBOZYME ACTIVITY - QUANTITATION OF INTERDOMAIN BINDING AND REACTION-RATE

Ribozyme kinetics and binding studies of a two-piece group II intron were used to mechanistically characterize a reaction analogous to the first step of RNA splicing. Domain 5 RNA (D5) catalyzes specific hydrolysis of an RNA substrate (exD123) composed of sequences surrounding the 5' exon/intron boundary. Both single- and multiple-turnover kinetic analyses produced similar values of k(cat) (0.04 and 0.1 min(-1), respectively) and K-m (270 and 190 nM, respectively) for 5' splice site hydrolysis catalyzed by D5. Base pairing is not believed to stabilize the binding of D5 to exD123, so the low K-m values suggest that unusual tertiary interactions provide considerable energetic stabilization to this complex. The strength of D5-exD123 binding was confirmed using a new direct binding assay based on gel filtration chromatography. In this initial application of the assay, which systematically underestimates binding by approximately 3-fold, K-d values were obtained in relative agreement with K-m. This agreement, together with agreement between kinetically determined variables, suggests that the reaction is described by a straightforward Michaelis-Menten mechanism and that k(cat) is the rate of the chemical step. This is supported by the log/linear pH/rate profile for k(cat) which has a slope 1 up to pH 6.2, consistent with a form of general base cataysis within this linear range. The shape of the plot suggests that the active site responsible for 5' splice site hydrolysis has a pK(a) of greater than or equal to 7.0.