METAL COORDINATION SITES THAT CONTRIBUTE TO STRUCTURE AND CATALYSIS IN THE GROUP-I INTRON FROM TETRAHYMENA

We have used nucleoside phosphorothioates (NTPalphaS) and a substitution-interference method to identify phosphate oxygens that appear to be important to guanosine cofactor addition in the self-splicing group I intron from Tetrahymena thermophila. For the majority of these phosphate oxygens, however, the effect of NTPalphaS substitution is significantly reduced in reactions containing the added presence of manganese ion (Mn2+) relative to magnesium ion (Mg2+) alone. The observed ''rescue'' of the NTPalphaS effect at these positions is thought to be due to the larger affinity of Mn2+ for sulfur. These data suggest the direct coordination of divalent metal ions within the highly conserved catalytic core of the Tetrahymena intron. Because many of these metal binding sites appear to be in positions of close backbone-backbone approach, and adjacent to the guanosine binding site the splice junction, we suggest roles for the corresponding ions in stabilizing tertiary structure and substrate recognition and as participants in catalysis.