RNA molecules and RNA-protein complexes are dynamic structures, and this motion is intrinsic to their function. The study of RNA folding mechanisms will provide physical insight into the assembly of RNAprotein complexes during gene regulation, and conformational changes that take place during catalysis. Although RNA secondary and tertiary interactions form quickly, some RNAs are easily trapped in misfolded states. Competition among alternative conformations can lead to disease if RNAs malfunction, or can be exploited for regulation of gene activity and replication of RNA viruses. Thus, the ability to understand and predict RNA function from its primary sequence is important to the analysis and treatment of genetic disease, and the development of therapeutic agents that target RNA molecules. The folding mechanism of a small group I ribozyme from Azoarcus will be compared with folding of the larger Tetrahymena ribozyme. This is an ideal system to study early steps in the assembly of RNA structure, because more than half the RNA folds within 100 ms, and the tertiary structure is stablilized by many types of metal ions. Hydroxyl radical footprinting using a synchrotron X-ray beam will be used to detect changes in RNA tertiary structure at 20 ms intervals. This method is unique, in that it resolves conformational changes at specific sites in the RNA. Stopped-flow fluorescence spectroscopy will be used to monitor global folding of RNAs containing 2-aminopurine and fluorescein, with 1 ms resolution. The proposed studies address how sequence and metal ion interactions direct the assembly of specific tertiary structures native conformations. Time resolved hydroxyl radical footprinting will also be used to study the assembly of RNA-protein complexes, using the bacterial RNase P holoenzyme as a test system. RNase P is an essential enzyme present in all cells. The folding pathway of the RNA subunit shares many properties with those of group I ribozymes, but it is not known how the protein subunit alters the formation of catalytically active RNA. As Xrays easily penetrate cells and whole tissues, this method will be adapted to study the three-dimensional structure of group I riboyzmes and RNase P in vivo.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Lewis, Catherine D
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Johns Hopkins University
Schools of Arts and Sciences
United States
Zip Code
Sharma, Indra Mani; Korman, Arthur; Woodson, Sarah A (2018) The Hfq chaperone helps the ribosome mature. EMBO J 37:
Sharma, Indra Mani; Rappé, Mollie C; Addepalli, Balasubrahmanyam et al. (2018) A metastable rRNA junction essential for bacterial 30S biogenesis. Nucleic Acids Res 46:5182-5194
Hao, Yumeng; Bohon, Jen; Hulscher, Ryan et al. (2018) Time-Resolved Hydroxyl Radical Footprinting of RNA with X-Rays. Curr Protoc Nucleic Acid Chem 73:e52
Abeysirigunawardena, Sanjaya C; Kim, Hajin; Lai, Jonathan et al. (2017) Evolution of protein-coupled RNA dynamics during hierarchical assembly of ribosomal complexes. Nat Commun 8:492
Hulscher, Ryan M; Bohon, Jen; Rappé, Mollie C et al. (2016) Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting. Methods 103:49-56
Lee, Hui-Ting; Kilburn, Duncan; Behrouzi, Reza et al. (2015) Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme. Nucleic Acids Res 43:1170-6
Abeysirigunawardena, Sanjaya C; Woodson, Sarah A (2015) Differential effects of ribosomal proteins and Mg2+ ions on a conformational switch during 30S ribosome 5'-domain assembly. RNA 21:1859-65
Kim, Hajin; Abeysirigunawarden, Sanjaya C; Chen, Ke et al. (2014) Protein-guided RNA dynamics during early ribosome assembly. Nature 506:334-8
Desai, Ravi; Kilburn, Duncan; Lee, Hui-Ting et al. (2014) Increased ribozyme activity in crowded solutions. J Biol Chem 289:2972-7
Hua, Boyang; Han, Kyu Young; Zhou, Ruobo et al. (2014) An improved surface passivation method for single-molecule studies. Nat Methods 11:1233-6

Showing the most recent 10 out of 32 publications