Published online 27 November 2005
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An obligate intermediate along the slow folding pathway of a group II intron ribozyme
1Department of Molecular Biophysics and Biochemistry, Yale University New Haven, CT 06520, USA 2Howard Hughes Medical Institute 266 Whitney Avenue, Box 208114 Yale University New Haven, CT 06520, USA
*To whom correspondence should be addressed. Tel: +1 203 432 5733; Fax: +1 203 432 5316; Email: anna.pyle{at}yale.edu
Received August 31, 2005. Revised November 4, 2005. Accepted November 4, 2005.
Most RNA molecules collapse rapidly and reach the native state through a pathway that contains numerous traps and unproductive intermediates. The D135 group II intron ribozyme is unusual in that it can fold slowly and directly to the native state, despite its large size and structural complexity. Here we use hydroxyl radical footprinting and native gel analysis to monitor the timescale of tertiary structure collapse and to detect the presence of obligate intermediates along the folding pathway of D135. We find that structural collapse and native folding of Domain 1 precede assembly of the entire ribozyme, indicating that D1 contains an on-pathway intermediate to folding of the D135 ribozyme. Subsequent docking of Domains 3 and 5, for which D1 provides a preorganized scaffold, appears to be very fast and independent of one another. In contrast to other RNAs, the D135 ribozyme undergoes slow tertiary collapse to a compacted state, with a rate constant that is also limited by the formation D1. These findings provide a new paradigm for RNA folding and they underscore the diversity of RNA biophysical behaviors.
Present address: Linhui Julie Su, Whitehead Institute for Biomedical Research, Cambridge, MA, USA
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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