Characterization of a local folding event of the Tetrahymena group I ribozyme: effects of oligonucleotide substrate length, pH, and temperature on the two substrate binding steps.

ABSTRACT

Binding of the Tetrahymena group I ribozyme's oligonucleotide substrate occurs in two steps: P1 duplex formation with the ribozyme's internal guide sequence which forms an "open complex" is followed by docking of the P1 duplex into tertiary interactions within the catalytic core which forms a "closed complex". By systematically varying substrate length, pH, and temperature, we have identified conditions under which P1 duplex formation, P1 docking, or the chemical cleavage step limits the rate of the ribozyme reaction. This has enabled characterization of the individual steps as a function of substrate length, pH, and temperature, leading to several conclusions. (1) The rate constant for formation of the open complex is largely independent of substrate length, pH, and temperature, analogous to that of duplex formation in solution. This extends previous results suggesting that open complex formation entails mainly secondary structure formation and strengthens the view that the second binding step, P1 docking, represents a simple transition from secondary to tertiary structure in the context of an otherwise folded RNA. (2) The temperature dependence of the rate constant for P1 docking yields a negative activation entropy, in contrast to the positive entropy change previously observed for the docking equilibrium. This suggests a model in which tertiary interactions are not substantially formed in the transition state for P1 docking. (3) Shortening the substrate by three residues decreases the equilibrium constant for P1 docking by 200-fold, suggesting that the rigidity imposed by full-length duplex formation facilitates formation of tertiary interactions. (4) Once docked, shortened substrates are cleaved at rates within 3-fold of that for the full-length substrate. Thus, all the active site interactions required to accelerate the chemical cleavage event are maintained with shorter substrates. (5) The rate constant of approximately 10(3) min(-1) obtained for P1 docking is significantly faster than the other steps previously identified in the tertiary folding of this RNA. Nevertheless, P1 docking presumably follows other tertiary folding steps because the P1 duplex docks into a core that is formed only upon folding of the rest of the ribozyme.