Fast formation of the P3-P7 pseudoknot: A strategy for efficient folding of the catalytically active ribozyme

Abstract

Formation of the P3-P7 pseudoknot structure, the core of group I ribozymes, requires long-range base pairing. Study of the Tetrahymena ribozyme appreciates the hierarchical folding of the large, multidomain RNA, in which the P3-P7 core folds significantly slower than do the other domains. Here we explored the formation of the P3-P7 pseudoknot of the Candida ribozyme that has been reported to concertedly fold to the catalytically active structure with a rate constant of 2 min −1 . We demonstrate that pseudoknot formation occurs during the rapid ribozyme compaction, coincident with formation of many tertiary interactions of the ribozyme. A low physiological concentration of magnesium (1.5 mM) is sufficient to fully support the pseudoknot formation. The presence of nonnative intermediates containing an unfolded P3-P7 region is evident. However, catalysis-based analysis shows these nonnative intermediates are stable and fail to convert to the catalytically active structure, suggesting that rapid pseudoknot formation is essential for folding of the active ribozyme. Interestingly, RNAstructure predicts no stable Alt P3 structure for the Candida ribozyme, but two stable Alt P3s for the Tetrahymena ribozyme, explaining the dramatic difference in folding of the P3-P7 core of these two ribozymes. We propose that rapid formation of the P3-P7 pseudoknot represents a folding strategy ensuring efficient production of the catalytically active structure of group I ribozymes, which sheds new light on the mechanism of effective ribozyme folding in vivo.

RNA Tertiary Interactions Mediate Native Collapse of a Bacterial Group I Ribozyme

“…43,44 By contrast, a similar mispairing is not predicted to form in the wild-type Azoarcus ribozyme. 20,45 Taken together, the results presented here argue that Mg 2C -induced collapse of the Azoarcus group I ribozyme produces native-like intermediates that are stabilized by tertiary interactions, as previously suggested for this RNA 21 and another group I ribozyme. 18 In turn, this suggests that native interactions within the RNA nucleate the collapse transition.…”

Slow formation of a pseudoknot structure is rate limiting in the productive co-transcriptional folding of the self-splicing Candida intron

“…Consistently, our study of the Candida group I ribozyme shows that refolding of the P3-P7 pseudoknot in the productive folding pathway is fast and not rate limiting, and the failure of rapid P3-P7 formation leads the ribozyme to a nonproductive folding pathway (Xiao et al 2003;Zhang et al 2005; this study). A study from the Woodson group also shows that refolding of the P3-P7 of the Azoarcus ribozyme is much more rapid than folding of the catalytically active ribozyme (Rangan et al 2003).…”

“…Co-transcriptional folding of the self-splicing Candida intron is twice as fast as refolding When the purified transacting Candida ribozyme Ca.L-11 that catalyzes the esterification reaction at the 59 splice site is refolded in the presence of Mg 2+ $2 mM, the major ribozyme population reaches its active structure with a rate constant of 2 min À1 via a pathway in which the P3-P7 pseudoknot forms much more rapidly (Xiao et al 2003;Zhang et al 2005). Another transacting Candida ribozyme catalyzing the exon ligation at 39 splice site also shows a fast folding constant ($1.8 min…”

Predicting the secondary structures and tertiary interactions of 211 group I introns in IE subgroup

“…Our study of the folding of a group I intron (subgroup IE) from the 26S rRNA gene of Candida albicans has demonstrated essential differences between folding of this group I ribozyme and that of the Tetrahymena group I ribozyme. This difference is attributed to the substantial differences in the peripheral interactions and in the tendency of mispairing of the P3 stem of those two introns (28–30). Consistently, an IC3 intron Azoarcus group I intron sharing a similar core sequence, but containing very different peripheral interactions from the Tetrahymena intron, folds very fast to the native structure, in contrast to the slow folding of the Tetrahymena intron (8,24–26,31).…”