GUANOSINE BINDS TO THE TETRAHYMENA RIBOZYME IN MORE THAN ONE-STEP, AND ITS 2'-OH AND THE NONBRIDGING PRO-S-P PHOSPHORYL OXYGEN AT THE CLEAVAGE SITE ARE REQUIRED FOR PRODUCTIVE DOCKING

The dynamics of binding of various guanosine, or G, substrates to the Tetrahymena thermophila L-21 ScaI ribozyme have been investigated by f luorescence-detected stopped-flow experiments. Upon rapid mixing of va rious G substrates with a preformed complex of the ribozyme and the fl uorescent 5' splice site analogue CCUCU epsilon A, fluorescence transi ents that provide rates for binding of G substrates and the rate-limit ing step for transesterification are observed. The measured apparent b imolecular rate constant for binding of pG is 10(3) M-1 s(-1), much sl ower than expected for diffusion. pG appears to bind to the preformed complex of the ribozyme and CCUCU epsilon A in at least two steps, a b imolecular step followed by at least one conformational change. This t wo-step binding of pG, involving a rapid pre-equilibrium, leads to the slow apparent rate constant for binding of pG. Furthermore, the 2'-OH of pG and of the 3' terminal G of the G substrate GUCG and the nonbri dging pro-S-p phosphoryl oxygen atom at the site of phosphoryl transfe r on CCUCU epsilon A appear to mediate formation of a properly conform ed docked ternary complex of the G substrate, 5' splice site, and ribo zyme which may represent an intermediate required for initiation of tr ansesterification. It is possible that the 2'-OH of pG and this nonbri dging pro-S-p phosphoryl oxygen interact, directly or indirectly, with one another.