Concerted folding of a Candida ribozyme into the catalytically active structure posterior to a rapid RNA compaction

Abstract

Folding of the major population of Tetrahymena intron RNA into the catalytically active structure is trapped in a slow pathway. In this report, folding of Candida albicans intron was investigated using the trans-acting Ca.L-11 ribozyme as a model. We demonstrated that both the catalytic activity (k(obs)) and compact folding equilibrium of Ca.L-11 are strongly dependent on Mg(2+) at physiological concentrations, with both showing an Mg(2+) Hill coefficient of 3. Formation of the compact structure of Ca.L-11 is shown to occur very rapidly, on a subsecond time scale similar to that of RNase T1 cleavage. Most of the ribozyme RNA population folds into the catalytically active structure with a rate constant of 2 min(-1) at 10 mM Mg(2+); neither slower kinetics nor obvious Mg(2+) inhibition is observed. These results suggest that folding of the Ca.L-11 ribozyme is initiated by a rapid magnesium-dependent RNA compaction, which is followed by a slower searching for the native contacts to form the catalytically active structure without interference from the long-lived trapped states. This model thus provides an ideal system to address a range of interesting aspects of RNA folding, such as conformational searching, ion binding and the role of productive intermediates.

Monovalent cations use multiple mechanisms to resolve ribozyme misfolding

“…2B). Apparently, substrate cleavage was a much faster process than the ribozyme folding (2 min -1 ) (Xiao et al 2003). We thus validated that the observed ribozyme activity (k obs ) in the present study reflects the rate of ribozyme folding.…”

“…Although folding of group I introns shares some common mechanisms in responding to magnesium (Woodson 2005), folding of the Candida ribozyme, a group I ribozyme derived from the 26S rRNA gene of the opportunistic fungal pathogen Candida albicans, behaves quite differently from that of the Tetrahymena ribozyme in many aspects (Sclavi et al 1998;Zhuang et al 2000;Xiao et al 2003;Zhang et al 2005). In this report, we studied independent functions of monovalent cations in folding of a trans-acting Candida ribozyme.…”

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).…”

“…2C,E), suggesting a faster ribozyme refolding than the AON binding. This is consistent with the fast refolding of the active trans-acting Candida ribozymes (Xiao et al 2003;Bao et al 2008). Strikingly, the co-transcriptional ribozyme folding was dramatically slowed by AONs targeting two pseudoknot structures, including AONs 239-260R, 239-262R, 242-260R, and 242-262R that target the P3-P7 pseudoknot, as well as AON 1-26R targeting the P1-P10 pseudoknot (Fig.…”

“…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…”