In addition, earlier studies suggested that the presence of a bifurcated aspartate bridging the two active site Mg 2+ ions was essential for RNase activity and that release of the frustration of an initially undercoordinated Mg 2+ in site B played a key role in catalysis. As these two features are absent from the experimentally reconstructed structure of the M5:rRNA complex, this was an opportunity to re-examine their role in RNase catalysis.