Conformational rearrangements in the catalytic mechanism of the Tetrahymena ribozyme: Influence of substrate structure,pH, and divalent cations

Conformational dynamics in catalysis by the Tetrahymena thermophila L-21 Sca I ribozyme have been investigated by fluorescence-detected stopped-flow experiments utilizing the fluorescent 5' splice site analogue CCUCUepsilonA.;Analysis of fluorescence transients observed upon rapid mixing of various G substrates with a pre-formed complex of ribozyme and CCUCUepsilonA reveals that pG appears to bind in two steps, a bimolecular step followed by a conformational change. This two step binding of pG, involving a rapid pre-equilibrium, leads to the slow apparent rate constant, 103 M-1s -1, for binding of pG. Furthermore, the 2' OH of pG and the non-bridging pro-Sp phosphoryl oxygen atom at the site of phosphoryltransfer on CCUCUepsilonA appear to mediate formation of a properly conformed docked ternary complex of G substrate, 5' splice site, and ribozyme which may represent an intermediate required for initiation of transesterification. It is possible that the 2' OH of pG and this non-bridging pro-Sp phosphoryl oxygen interact, directly or indirectly, with one another.;The effects of pH variation on observed rate constants for steps in the catalytic pathway of the Tetrahymena thermophila ribozyme have been examined. Observed rates for cleavage detected with 32P labelled substrates and observed rate constants for fluorescence transients after pG docking are shown to exhibit differing and unexpected dependencies on pH at 5 mM Mg2+ and 15°C, suggesting that complex conformational changes underlie the catalytic mechanism of the ribozyme.;Observed rate constants for steps in the catalytic pathway of the Tetrahymena thermophila ribozyme have been measured in the presence and absence of Mn2+, as a supplement to Mg2+. Mn2+ appears to alter the dynamics of both pG docking and the phosphoryltransfer step. Results obtained as a function of introducing Mn 2+ to the experimental system at different times relative to renaturation of ribozyme and addition of substrates suggest that specific binding of divalent metal ions to the RNA is fast compared to rates of detected rearrangements along the catalytic pathway. Mn2+ is demonstrated to influence pG docking likely by mediating specific local structure at or near the site of docking, possibly involving uptake of Mn2+ during pG docking.