The 2′-OH group at the group II intron terminus acts as a proton shuttle

Group II introns are self-splicing ribozymes that excise themselves from precursor RNAs and catalyze the joining of flanking exons. Excised introns can behave as parasitic RNA molecules, catalyzing their own insertion into DNA and RNA via a reverse-splicing reaction. Previous studies have identified mechanistic roles for various functional groups located in the catalytic core of the intron and within target molecules. Here we introduce a new method for synthesizing long RNA molecules with a modified nucleotide at the 3′-terminus. This modification allows us to examine the mechanistic role of functional groups adjacent to the reaction nucleophile. During reverse-splicing, the 3′-OH group of the intron terminus attacks the phosphodiester linkage of spliced exon sequences. Here we show that the adjacent 2′-OH group on the intron terminus plays an essential role in activating the nucleophile by stripping away a proton from the 3′-OH and then shuttling it from the active-site.