Figure 4.

(a) The extended and tertiary-folded secondary structures of tP5abc. A magnesium ion core forms on tertiary folding and causes extensive rearrangement of P5c and the adenine-rich bulge (15). Each solid bar denotes a Watson–Crick base pair, and each hollow bar denotes a non-Watson–Crick base pair. The green dots in the folded form represent five magnesium ions revealed in the crystal of P5-P6 that directly coordinate (thick green lines) to G or phosphate oxygen (p), or indirectly coordinate to phosphate mediated by a water molecule (thin green lines). Mutations (colored in blue) enable the catalytic core of a group I intron to become protected faster from RNase H digestion during magnesium-ion induced folding (11). These mutations do not disrupt the stability of an extended P5abc (or tP5abc): A186U, A183U, and U167C do not disrupt the base pairing; A171G converts the GCAA tetraloop into a GCGA tetraloop, which belongs to the same GNRA tetraloop family and should have similar structure and stability (33). NMR experiments showed that mutant A186U of tP5abc folds into a similar secondary structure as the wild-type tP5abc and that it does not fold in the presence of Mg²⁺ (Fig. 3). Mutant + 174G is an insertion mutant that can form a stable GNAAA 5-nt loop that is analogous to a GNRA tetraloop (38). (b) Comparison of the extended tP5abc with the tertiary folded tP5abc in the P4-P6 crystal. The folded tP5abc is generated by removing four base pairs in the P5b stem from the native structure of P4-P6 (34). The folded tP5abc is highly compact at the 3-helix junction. The extended tP5abc generated from NMR constraints was the lowest energy structure obtained from one hundred random starting structures.