TY - JOUR
T1 - An alternative route for the folding of large RNAs
T2 - Apparent two-state folding by a group II intron ribozyme
AU - Su, Linhui Julie
AU - Brenowitz, Michael
AU - Pyle, Anna Marie
N1 - Funding Information:
The authors thank Tobin Sosnick, Art Palmer, Eric Gouaux, and Andrew Miranker for insightful advice and suggestions, Peter Moore for suggesting hydrodynamic techniques for studying group II introns, Alex de Lencastre and Christina Waldsich for helpful discussions on the manuscript, and Rich Olson for advice on analytical ultracentrifugation. Funding for the Jasco J810 Spectropolarimeter was obtained from NIH grant GM34509 to Koji Nakanishi. Funding for the Beckman XL-I centrifuges were obtained from NIH shared instrumentation grant S10 RR12848 to Columbia University and GM54160 to Donald Engelman (Yale University). We acknowledge funding from NIH to L.J.S. (Biophysics Training Grant T32 GM08281), M.B. (GM52348), and A.M.P. (RO1 GM50313). A.M.P. is an Investigator of the Howard Hughes Medical Institute.
PY - 2003/12/5
Y1 - 2003/12/5
N2 - Despite a growing literature on the folding of RNA, our understanding of tertiary folding in large RNAs derives from studies on a small set of molecular examples, with primary focus on group I introns and RNase P RNA. To broaden the scope of RNA folding models and to better understand group II intron function, we have examined the tertiary folding of a ribozyme (D135) that is derived from the self-splicing ai5γ intron from yeast mitochondria. The D135 ribozyme folds homogeneously and cooperatively into a compact, well-defined tertiary structure that includes all regions critical for active-site organization and substrate recognition. When D135 was treated with increasing concentrations of Mg2+ and then subjected to hydroxyl radical footprinting, similar Mg2+ dependencies were seen for internalization of all regions of the molecule, suggesting a highly cooperative folding behavior. In this work, we show that global folding and compaction of the molecule have the same magnesium dependence as the local folding previously observed. Furthermore, urea denaturation studies indicate highly cooperative unfolding of the ribozyme that is governed by thermodynamic parameters similar to those for forward folding. In fact, D135 folds homogeneously and cooperatively from the unfolded state to its native, active structure, thereby demonstrating functional reversibility in RNA folding. Taken together, the data are consistent with two-state folding of the D135 ribozyme, which is surprising given the size and multi-domain structure of the RNA. The findings establish that the accumulation of stable intermediates prior to formation of the native state is not a universal feature of RNA folding and that there is an alternative paradigm in which the folding landscape is relatively smooth, lacking rugged features that obstruct folding to the native state.
AB - Despite a growing literature on the folding of RNA, our understanding of tertiary folding in large RNAs derives from studies on a small set of molecular examples, with primary focus on group I introns and RNase P RNA. To broaden the scope of RNA folding models and to better understand group II intron function, we have examined the tertiary folding of a ribozyme (D135) that is derived from the self-splicing ai5γ intron from yeast mitochondria. The D135 ribozyme folds homogeneously and cooperatively into a compact, well-defined tertiary structure that includes all regions critical for active-site organization and substrate recognition. When D135 was treated with increasing concentrations of Mg2+ and then subjected to hydroxyl radical footprinting, similar Mg2+ dependencies were seen for internalization of all regions of the molecule, suggesting a highly cooperative folding behavior. In this work, we show that global folding and compaction of the molecule have the same magnesium dependence as the local folding previously observed. Furthermore, urea denaturation studies indicate highly cooperative unfolding of the ribozyme that is governed by thermodynamic parameters similar to those for forward folding. In fact, D135 folds homogeneously and cooperatively from the unfolded state to its native, active structure, thereby demonstrating functional reversibility in RNA folding. Taken together, the data are consistent with two-state folding of the D135 ribozyme, which is surprising given the size and multi-domain structure of the RNA. The findings establish that the accumulation of stable intermediates prior to formation of the native state is not a universal feature of RNA folding and that there is an alternative paradigm in which the folding landscape is relatively smooth, lacking rugged features that obstruct folding to the native state.
KW - Group II intron
KW - RNA folding
KW - Ribozyme
KW - Splicing
KW - Tertiary structure
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U2 - 10.1016/j.jmb.2003.09.071
DO - 10.1016/j.jmb.2003.09.071
M3 - Article
C2 - 14636593
AN - SCOPUS:0242636400
SN - 0022-2836
VL - 334
SP - 639
EP - 652
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 4
ER -