Multistage collapse of a bacterial ribozyme observed by time-resolved small-angle X-ray scattering.
Ribozymes must fold into compact, native structures to function properly in the cell. The first step in forming the RNA tertiary structure is the neutralization of the phosphate charge by cations, followed by collapse of the unfolded molecules into more compact structures. The specificity of the collapse transition determines the structures of the folding intermediates and the folding time to the native state. However, the forces that enable specific collapse in RNA are not understood. Using time-resolved SAXS, we report that upon addition of 5 mM Mg(2+) to the Azoarcus group I ribozyme up to 80% of chains form compact structures in less than 1 ms. In 1 mM Mg(2+), the collapse transition produces extended structures that slowly approach the folded state, while > or = 1.5 mM Mg(2+) leads to an ensemble of random coils that fold with multistage kinetics. Increased flexibility of molecules in the intermediate ensemble correlates with a Mg(2+)-dependent increase in the fast folding population and a previously unobserved crossover in the collapse kinetics. Partial denaturation of the unfolded RNA with urea also increases the fraction of chains following the fast-folding pathway. These results demonstrate that the preferred collapse mechanism depends on the extent of Mg(2+)-dependent charge neutralization and that non-native interactions within the unfolded ensemble contribute to the heterogeneity of the ribozyme folding pathways at the very earliest stages of tertiary structure formation.
Roh
Joon Ho
JH
Department of Materials Science and Engineering, University of Maryland, College Park, Maryland 20742, USA. rohmio1973@gmail.com
Guo
Liang
L
Kilburn
J Duncan
JD
Briber
Robert M
RM
Irving
Thomas
T
Woodson
Sarah A
SA
D020627:Azoarcus / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009690:Nucleic Acid Conformation / Q000187:drug effects; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D053838:Scattering, Small Angle; D013997:Time Factors; D014961:X-Ray Diffraction
D016337:RNA, Catalytic; D008274:Magnesium