Effects of substrate structure on the kinetics of circle opening reactions of the self-splicing intervening sequence from Tetrahymena thermophila: Evidence for substrate and Mg²⁺ binding lnteractions

The self-splicing intervening sequence from the precursor rRNA of Tetrahymena thermophila cyclizes to form a covalently closed circle. This circle can be reopened by reaction with oligonucleotides or water. The kinetics of circle opening as a function of substrate and Mg²⁺ concentrations have been measured for dCrU, rCdU, dCdT, and H₂O addition. Comparisons with previous results for rCrU suggest: (1) the 2' OH of the 5' sugar of a dinucleoside phosphate is involved in substrate binding, and (2) the 2' OH of the 3' sugar of a dimer substrate is involved in Mg²⁺ binding. Evidently, the binding site for a required Mg ion is dependent on both the ribozyme and the dimer substrate.The apparent activation energy and entropy for circle opening by hydrolysis are 31 kcal/mol and 50 eu, respectively. The large, positive activation entropy suggests a partial unfolding of the ribozyme is required for reaction.