Folding mechanism of the Tetrahymena ribozyme P4-P6 domain

Synchrotron X-ray-dependent hydroxyl radical footprinting was used to probe the folding kinetics of the P4-P6 domain of the Tetrahymena group I ribozy me, which forms a stable, closely packed tertiary structure. The 160-nt dom ain folds independently at a similar rate (similar to 2 s(-1)) as it does i n the ribozyme, when folding is measured in 10 mM sodium cacodylate and 10 mM MgCl2. Surprisingly, tertiary interactions around a three-helix junction (P5abc) within the P4-P6 domain fold at least 25 times more rapidly (k gre ater than or equal to 50 s(-1)) in isolation, than when part of the wild-ty pe P4-P6 RNA. This difference implies that long-range interactions in the P 4-P6 domain can interfere with folding of P5abc. P4-P6 was observed to fold much faster at higher ionic strength than in 10 mM sodium cacodylate. Anal ytical centrifugation was used to measure the sedimentation and diffusion c oefficients of the unfolded RNA. The hydrodynamic radius of the RNA decreas ed from 58 To 46 Angstrom over the range of 0-100 mM NaCl. We propose that at low ionic strength, the addition of Mg2+ causes the domain to collapse t o a compact intermediate where P5abc is trapped in a non-native structure. At high ionic strength, the RNA rapidly collapses to the native structure. Faster folding most likely results from a different average initial conform ation of the RNA in higher salt conditions.