Exploration of the transition state for tertiary structure formation between an RNA helix and a large structured RNA.
Docking of the P1 duplex into the pre-folded core of the Tetrahymena group I ribozyme exemplifies the formation of tertiary interactions in the context of a complex, structured RNA. We have applied Phi-analysis to P1 docking, which compares the effects of modifications on the rate constant for docking (k(dock)) with the effects on the docking equilibrium (K(dock)). To accomplish this we used a single molecule fluorescence resonance energy transfer assay that allows direct determination of the rate constants for formation of thermodynamically favorable, as well as unfavorable, states. Modification of the eight groups of the P1 duplex that make tertiary interactions with the core and changes in solution conditions decrease K(dock) up to 500-fold, whereas k(dock) changes by </=2-fold. The absence of effects on k(dock), both from atomic modifications and global perturbations, strongly suggests that the transition state for docking is early and does not closely resemble the docked state. These results, the slow rate of docking of 3s(-1), and the observation that a modification that is expected to increase the degrees of freedom between the P1 duplex and the ribozyme core accelerates docking, suggest a model in which a kinetic trap(s) slows docking substantially. Nonetheless, urea does not increase k(dock), suggesting that there is little change in the exposed surface area between the trapped, undocked state and the transition state. The findings highlight that urea and temperature dependencies can be inadequate to diagnose the presence of kinetic traps in a folding process. The results described here, combined with previous work, provide an in-depth view of an RNA tertiary structure formation event and suggest that large, highly structured RNAs may have local regions that are misordered.
Department of Biochemistry, B400 Beckman Center, Stanford University, Stanford, CA 94305-5307, USA.
Laura E
LE
Bartley
Xiaowei
X
Zhuang
Rhiju
R
Das
Steven
S
Chu
Daniel
D
Herschlag
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016053:RNA, Protozoan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013768:Tetrahymena / Q000201:enzymology / Q000235:genetics; D013816:Thermodynamics
D016337:RNA, Catalytic; D016053:RNA, Protozoan