Kinetic pathway for folding of the Tetrahymena ribozyme revealed by three UV-inducible crosslinks.
The kinetics of RNA folding were examined in the L-21 ribozyme, an RNA enzyme derived from the self-splicing Tetrahymena intron. Three UV-inducible crosslinks were mapped, characterized, and used as indicators for the folded state of the ribozyme. Together these data suggest that final structures are adopted first by the P4-P6 independently folding domain and only later in a region that positions the P1 helix (including the 5' splice site), a region whose folding is linked to that of a portion of the catalytic core. At intermediate times, a non-native structure forms in the region of the triple helical scaffold, which connects the major folding domains. At 30 degrees C, the unfolded ribozyme passes through these stages with a half-life of 2 min from the time magnesium cations are provided. At higher temperatures, the half-life is shortened but the order of events is unchanged. Thermal melting of the fully folded ribozyme also revealed a multi-stage process in which the steps of folding are reversed: the kinetically slowest structure is the least stable and melts first. These structures of the ribozyme also bind Mg2+ cooperatively and their relative affinity for binding seems to be a major determinant in the order of events during folding. Na+ can also substitute for Mg2+ to give rise to the same crosslinkable structures, but only at much higher concentrations. Specific binding sites for Mg2+ may make this cation particularly efficient at electrostatic stabilization during folding of these ribozyme structures.
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Department of Molecular, Cellular and Developmental Biology, Howard Hughes Medical Institute, University of Colorado, Boulder 80309-0215, USA. wdowns@fred.fhcrc.org
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D000818:Animals; D001483:Base Sequence; D003432:Cross-Linking Reagents; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D016053:RNA, Protozoan / Q000737:chemistry / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D013696:Temperature; D013768:Tetrahymena / Q000235:genetics*; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D016337:RNA, Catalytic; D016053:RNA, Protozoan; D012964:Sodium; D008274:Magnesium