title: Folding pathways of the Tetrahymena ribozyme.
abstract: Like many structured RNAs, the Tetrahymena group I intron ribozyme folds through multiple pathways and intermediates. Under standard conditions in vitro, a small fraction reaches the native state (N) with kobs ≈ 0.6 min(-1), while the remainder forms a long-lived misfolded conformation (M) thought to differ in topology. These alternative outcomes reflect a pathway that branches late in folding, after disruption of a trapped intermediate (Itrap). Here we use catalytic activity to probe the folding transitions from Itrap to the native and misfolded states. We show that mutations predicted to weaken the core helix P3 do not increase the rate of folding from Itrap but they increase the fraction that reaches the native state rather than forming the misfolded state. Thus, P3 is disrupted during folding to the native state but not to the misfolded state, and P3 disruption occurs after the rate-limiting step. Interestingly, P3-strengthening mutants also increase native folding. Additional experiments show that these mutants are rapidly committed to folding to the native state, although they reach the native state with approximately the same rate constant as the wild-type ribozyme (~1 min(-1)). Thus, the P3-strengthening mutants populate a distinct pathway that includes at least one intermediate but avoids the M state, most likely because P3 and the correct topology are formed early. Our results highlight multiple pathways in RNA folding and illustrate how kinetic competitions between rapid events can have long-lasting effects because the "choice" is enforced by energy barriers that grow larger as folding progresses.
authors.affiliation: Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
authors.forename: David
authors.initials: D
authors.lastname: Mitchell
authors.affiliation: Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA. Electronic address: rick_russell@cm.utexas.edu.
authors.forename: Rick
authors.initials: R
authors.lastname: Russell
mesh_terms: D020029:Base Pairing; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D059370:RNA Folding; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D013768:Tetrahymena / Q000737:chemistry* / Q000201:enzymology*
chemical_list: D016337:RNA, Catalytic
keywords: RNA folding; RNA topology; catalytic RNA; group I intron; structured RNA
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