The tertiary structure of the hairpin ribozyme is formed through a slow conformational search.
The hairpin ribozyme is a small catalytic RNA comprised of two internal loops carried on two adjacent arms of a four-way helical junction (4WJ). To achieve catalytic activity, the ribozyme folds into a compact conformation that facilitates the formation of tertiary interactions between the two loops. We have investigated the folding kinetics of the natural 4WJ form of the hairpin ribozyme, as well as a minimal construct consisting of just the two loop-containing duplexes, by means of stopped-flow fluorescence resonance energy transfer between donor and acceptor probes attached to the ends of the loop-bearing arms. Folding was initiated by the addition of Mg(2+) ions or a pseudosubstrate strand to the ribozyme, and the ensuing changes in the emission of both donor and acceptor were monitored over time. Both ribozyme constructs exhibited slow, biphasic kinetic behavior, attributed to two parallel folding pathways leading to compact, docked structures. Two distinct folding rates were observed across a range of Mg(2+) concentrations, and increasing amounts of Mg(2+) accelerated both rates. Notably, both rates were essentially independent of temperature, indicating that the corresponding activation enthalpies were negligible, in contrast to the large activation enthalpies generally observed for RNA folding processes. Instead, the slow folding was due to unfavorable entropy changes in reaching the transition state, indicating that the ribozyme tertiary structure forms through a slow conformational search. These features were observed in both forms of the ribozyme, indicating that the conformational search is confined to the two loop regions and is largely independent of the overall ribozyme architecture. Conformational search may be a general mechanism of tertiary structure formation in RNA.
Pljevaljcić
Goran
G
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Klostermeier
Dagmar
D
Millar
David P
DP
D002413:Cations, Divalent; D019793:Fluorescein / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry; D013696:Temperature
D002413:Cations, Divalent; D005456:Fluorescent Dyes; D016337:RNA, Catalytic; D012235:Rhodamines; C418481:hairpin ribozyme; C005358:tetramethylrhodamine; D008274:Magnesium; D019793:Fluorescein