Metal ion coordination to 2′ functionality of guanosine mediates substrate–guanosine coupling in group I ribozymes: implications for conserved role of metal ions and for variability in RNA folding in ribozyme catalysis

Divalent metal ions are necessary in the self splicing reaction of group I introns, and we report that metal interaction to the 2′ position of guanosine for the Azoarcus ribozyme is required for catalysis. Moreover, this metal coordination promotes the guanosine–substrate coupled binding to the ribozyme, which is another conserved feature seen across phylogenetic boundaries. Typically there is a 4–9-fold difference in binding of G to Efree versus E · S. In the Tetrahymena ribozyme’s case this substrate–guanosine communication was attributed to conformational change(s) that lead to cooperative binding of the two cofactors which is almost nonexistent at low temperatures (4 °C). In the prokaryotic Azoarcus ribozyme we also see a 4–5-fold difference in binding of the guanosine/substrate to Efree versus E · G or E · S at 10 °C that is attributed to guanosine–substrate coupling. This coupling is diminished when the metal (Mg2+) coordination to the 2′ is disrupted with use of 2′-amino-2′-deoxyguanosine. The coupling is restored when softer Mn2+ ions are added to the buffer. This evidence generalizes a model for group I ribozyme catalysis that involves metal coordination to the 2′ position of guanosine. However, we see one striking difference in that the guanosine–substrate coupling is reversed. In the Azoarcus system (10 °C) the guanosine/substrate binds 5-fold more tightly to Efree than to E · S or E · G, which is the opposite for Tetrahymena even when the later is run at 4 °C. One implication for this difference in coupling is that the Azoarcus is in a folded state well accommodated for guanosine or substrate binding. This initial binding actually causes a conformational change that retards the subsequent binding of the second cofactor, which contrasts what was found for the Tetrahymena ribozyme. These results indicate that while the role for the metal ions in the chemical catalysis is conserved across phylogenetic boundaries, there is variability in the folding pattern of the ribozyme that leads to phosphoryl transfer.