The goal of our research is to understand how RNA folds into biologically active structures, and how the mechanisms of RNA folding determine the assembly of RNA-protein complexes. RNA molecules play a role in nearly every aspect of gene expression, including transcription, splicing, protein synthesis, and replication. In order to perform these functions, RNA sequences fold into specific three-dimensional structures. As is usually true for proteins, RNA structures are dynamic, undergoing conformational shifts in response to substrates or regulators. Catalytic RNAs or ribozymes are wonderful systems in which to study the RNA folding problem, because the activity of the native RNA is easy to measure. We are using group I introns or ribozymes from Tetrahymena thermophila (a unicellular ciliated protozoan) and Azoarcus sp. (a nitrogen fixing bacterium) to study the mechanisms of RNA folding. In nature, the Tetrahymena intron RNA catalyzes its own excision from the pre-ribosomal RNA, and joins the two rRNA exons of the precursor back together. In vitro, the self-splicing reaction requires only the RNA, buffer, Mg2+, and a GTP cofactor. In collaboration with Michael Brenowitz and Mark Chance at Albert Einstein College of Medicine, we have developed a synchrotron "X-ray footprinting" method to probe the tertiary structure of RNA with millisecond time resolution. Chemical reagents, such as hydroxyl radical, selectively cleave regions of the RNA that are on the exterior of the folded structure. Irradiation with a synchrotron X-ray beam at the National Synchrotron Light Source at Brookhaven National Lab generates enough hydroxyl radical to cleave the RNA in 10 ms, making it possible to take "snapshots" of the RNA structure as it forms. Using X-ray footprinting and other methods, we found that tertiary structure in the P4-P6 domain of the Tetrahymena ribozyme folds in 1-2 seconds, while the domain containing P3 and P7 folds very slowly (1-60 minutes). These partially misfolded intermediates are stable and long-lived, because they must partially unfold before rearranging to the catalytically active (or native) conformation. A variety of methods are needed to understand the folding mechanism of large RNAs. X-ray footprinting enables us to observe specific changes in the tertiary structure of the RNA on the millisecond time scale. Stopped-flow fluorescence spectroscopy monitors real-time changes in the global conformation of the RNA. Native polyacrylamide gel electrophoresis is a simple and direct way of separating intermediates. We are now using these methods to investigate the assembly mechanism of ribosomal RNA-protein complexes. Since many proteins recognize and stabilize specific conformations of the RNA, the assembly process is inextricably linked to folding of the RNA. Although some steps in RNA folding occur very slowly in vitro, group I introns fold and splice rapidly in the cell. Proteins may enhance splicing either by binding and stabilizing the active conformation of the intron, or by increasing the folding rate. Plasmid reporter assays are being used to identify proteins that increase the rate of splicing. Pan, J., Thirumalai, D. and Woodson, S. A. (1997) Folding of RNA involves parallel pathways. J. Mol. Biol. 273, 7-13. Sclavi, B., Sullivan, M., Chance, M. R., Brenowitz, M. and Woodson, S. A. (1998) Visualizing RNA folding at millisecond intervals with x-ray footprinting. Science 279, 1940-1943. Pan, J. and Woodson, S. A. (1998) Folding intermediates of a self-splicing RNA: mispairing of the catalytic core. J. Mol. Biol. 280, 597-609. Pan, J., Deras, M. L., and Woodson, S. A. (2000) Fast folding of a ribozyme by stabilizing core interactions: evidence for multiple folding pathways in RNA. J. Mol. Biol. 296, 133-144. Thirumalai, D., Lee, N., Woodson, S. A. and Klimov, D. K. (2001) Early events in RNA folding. Annu. Rev. Phys. Chem. 52, 751-762. Heilman-Miller, S. L., Thirumalai, D. and Woodson, S. A. (2001) Role of counterion condensation in folding of the Tetrahymena ribozyme. I. Equilibrium stabilization by cations. J. Mol. Biol. 306, 1157-1166. Jackson, S. A., Cannone, J. J., Lee, J. C., Gutell, . R. and Woodson, S. A. (2002) Distribution of rRNA introns in the three-dimensional structure of the ribosome. J. Mol. Biol. 323, 35-52. Rangan, P., Masquida, B., Westhof, E. and Woodson, S. A. (2003) Assembly of core helices and rapid tertiary folding of a small bacterial group I ribozyme. Proc. Natl. Acad. Sci. U.S.A. 100, 1574-1579. Heilman-Miller, S. L. and Woodson, S. A. (2003) Perturbed folding kinetics of circularly permuted RNAs with altered topology. J. Mol. Biol. 328: 385-394. Heilman-Miller, S. L. and Woodson, S. A. (2003) Effect of transcription on folding of the Tetrahymena ribozyme. RNA 9, 722-733. Rangan, P. and Woodson, S. A. (2003) Structural requirement for Mg2+ binding in the group I intron core. J. Mol. Biol. 329, 229-238. Perez-Salas, U. A., Rangan, P., Krueger, S., Briber, R. M., Thirumalai, D. and Woodson, S. A. (2004) Compaction of a bacterial group I ribozyme coincides with the assembly of core helices. Biochemistry 43, 1746-1753. Koculi, E., Lee, N.-K., Thirumalai, D. and Woodson, S. A. (2004) Folding of the Tetrahymena ribozyme by polyamines: importance of counterion valence and size. J. Mol. Biol. 341, 27-36. Koduvayur, S. and Woodson, S. A. (2004) Intracellular folding of the Tetrahymena group I intron depends on exon sequence and promoter choice. RNA 10, 1526-1532. Lease, R. A. and Woodson, S. A. (2004) Cycling of the Sm-like protein Hfq on the DsrA small regulatory RNA. J. Mol. Biol. 344, 1211-1123.
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