Abstract
The presence of non-conserved peripheral elements in all naturally occurring group I introns underline their importance in ensuring the natural intron function. Recently, we reported that some peripheral elements are conserved in group I introns of IE subgroup. Using self-splicing activity as a readout, our initial screening revealed that one such conserved peripheral elements, P2.1, is mainly required to fold the catalytically active structure of the Candida ribozyme, an IE intron. Unexpectedly, the essential function of P2.1 resides in a sequence-conserved short stem of P2.1 but not in a long-range interaction associated with the loop of P2.1 that stabilizes the ribozyme structure. The P2.1 stem is indispensable in folding the compact ribozyme core, most probably by forming a triple helical interaction with two core helices, P3 and P6. Surprisingly, although the ribozyme lacking the P2.1 stem renders a loosely folded core and the loss of self-splicing activity requires two consecutive transesterifications, the mutant ribozyme efficiently catalyzes the first transesterification reaction. These results suggest that the intron self-splicing demands much more ordered structure than does one independent transesterification, highlighting that the universally present peripheral elements achieve their functional importance by enabling the highly ordered structure through diverse tertiary interactions.