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

Binding of the Tetrahymena group I ribozyme's oligonucleotide substrate occ urs in two steps: P1 duplex formation with the ribozyme's internal guide se quence which forms an "open complex" is followed by docking of the P1 duple x into tertiary interactions within the catalytic core which forms a "close d 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. T his has enabled characterization of the individual steps as a function of s ubstrate length. pH, and temperature, leading to several conclusions. (1) T he rate constant for formation of the open complex is largely independent o f substrate length, pH, and temperature, analogous to that of duplex format ion 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 transit ion from secondary to tertiary structure in the context of an otherwise fol ded 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 mod el in which tertiary interactions are not substantially formed in the trans ition state for P1 docking. (3) Shortening the substrate by three residues decreases the equilibrium constant for P1 docking by 200-fold, suggesting t hat the rigidity imposed by full-length duplex formation facilitates format ion of tertiary interactions. (4) Once docked, shortened substrates are cle aved at rates within 3-fold of that for the full-length substrate. Thus, al l the active site interactions required to accelerate the chemical cleavage event are maintained with shorter substrates. (5) The rate constant of sim ilar to 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, pi docking presumably follows other tertiary folding steps be cause the Pi duplex docks into a core that is formed only upon folding of t he rest of the ribozyme.