METAL-ION DEPENDENCE OF ACTIVE-SITE STRUCTURE OF THE TETRAHYMENA RIBOZYME REVEALED BY SITE-SPECIFIC PHOTO-CROSS-LINKING

The L-21 ScaI ribozyme derived from the Tetrahymena group I intron recognizes its RNA substrate by base pairing, forming the P1 duplex, and cleaves it in a Mg2+-dependent transesterification reaction. In the presence of a substrate analog, photo-cross-linking of 5'-(azidophenacyl)-substituted L-21 ScaI ribozyme gives only two cross-linked products over a wide range of Mg2+ concentrations. Product 1 corresponds to the catalytically active RNA structure at high Mg2+ concentration, and product 3 to an inactive structure formed in the absence of Mg2+. The observed change in the ratio of cross-linked species 1 versus 3 as a function of Mg2+ concentration shows high cooperativity in the folding of the P1 duplex with the catalytic core of the ribozyme (Hill constant = 8 +/- 2). In addition, the site-specific photo-cross-linking detects for the first time a difference between the Mg2+-folded and the Ca2+-folded ribozyme structures, a difference which involves the positioning of P1. Introduction of cross-link 1 into the RNA does not render it catalytically active in the presence of Ca2+, consistent with Mg2+ being important in the chemical step in addition to its contribution to the correct positioning of P1.