The relationship between a primary sequence and the three-dimensional structure is a fundamental issue in macromolecular folding. RNA folding is governed by rules that represent an illuminating contrast to the more popular """"""""protein folding problem."""""""" RNA folding involves many metastable intermediates that are prerequisite for folding to the native structure. Previous studies, primarily ensemble measurements, demonstrated that elucidation of the structural and dynamic characteristics of these intermediates is crucial for understanding RNA folding. Ensemble measurements, however, measure average properties and often lack sufficient resolution to obtain detailed mechanistic insights involving folding intermediates. Because the ensemble generally populates several intermediate states under a variety of useful folding conditions, ensemble measurements only report an average behavior of these overlapping populations. This is clearly deleterious to; for example, establishing microscopic rates of reaction between the various states in the folding pathway. We will conduct single-molecule folding studies of two homologous ribozymes of abut 260 residues whose ensemble-determined folding pathways consist of defined, but overlapping intermediates and are free of kinetic traps. Folding will be monitored by fluorescence resonance energy transfer between two fluorophores attached at specific locations in the RNA.
Aim 1 is to characterize the distribution and structural fluctuations of folding intermediates and to compare them with structural fluctuations during folding.
Aim 2 is to identify fluctuations and dynamics among subdomains of the large ribozyme from Bacillus subtilis RNase P with strategic FRET labeling.
Aim 3 is to compare folding of mesophilic and thermophilic homologues. These results will elucidate folding pathways and their associate intermediates and address how sequence affects the energetics and structures of folding intermediates.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067961-04
Application #
7060736
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Lewis, Catherine D
Project Start
2003-05-01
Project End
2008-04-30
Budget Start
2006-05-01
Budget End
2008-04-30
Support Year
4
Fiscal Year
2006
Total Cost
$255,780
Indirect Cost
Name
University of Chicago
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
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