Ordered sequential intron splicing has to occur in certain pre-tRNAs harboring two introns. Pre-tRNA-Pro (GGG) of M. thermoautotrophicum contains two introns in the anticodon loop, one between nt 32 and 33 (32 nt in length) and the second one between nt 37 and 38 (16 nt in length). In the conventional cloverleaf scheme, bases 21–39 are replaced by asterisks. They are redrawn in an alternative 2D structure that fits the splicing motif requirement. The same lettering conventions as in Figures 1–4 are used. Only the hypothetical folding of the noncanonically located 32-nt intron is shown. In this pre-tRNA, the formation of the canonical splicing motif hBHBh′ (allowing the intron at position 32/33 to be spliced) obviously competes with the formation of the splicing motif corresponding to the intron located at the usual position 37/38 (boxed). Only after removal of the intron at position 32/33, can the intron–exon junction corresponding to intron 37/38 be folded into another hBHBh′ splicing motif (folding not shown).
