KINETICS AND THERMODYAMIC RNA FOLDING INTERMEDIATES MONITORED BY SAXS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. RNA, once thought to serve mainly as a courier of genetic information, is now known to play central structural and enzymatic roles in such complexes as the spliceosome and ribosome. To function, RNA must fold to a discrete three-dimensional structure, which introduces the RNA folding problem. Our previous SAXS work demonstrated that the Tetrahymena group I ribozyme undergoes a large, Mg2+-dependent collapse in folding from the denatured state to the folded state, and that collapse occurs substantially faster than overall folding. Further, we found that collapse is largely complete before any tertiary structure is observed to form, suggesting that a non-specifically collapsed state is populated early in folding. Here we propose to build on these observations by (i) obtaining structural information on the progression of intermediates that is populated after formation of the collapsed state; (ii) probing molecular features that affect the properties of the collapsed state; and (iii) monitoring the global structures of intermediates that are populated as the ribozyme is denatured by urea. These experiments are part of a larger project aimed at characterizing the folding behavior and the overall folding landscape for this RNA.