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The Skaggs Institute for Chemical Biology
Scientific Report 1999-2000

RNA Folding

J.R. Williamson, D. Abramovitz, S. Agalarov,* I. Baxter, T. Carlomagno, C.D. Cilley, K.T. Dayie, V. Feher, P. Funke, M. Hennig, D. Hoggan, J.W. Orr, Y. Pavlova, P.K. Radha, M. Recht, L.G. Scott, D.K. Treiber, M. Trevathan
* Russian Academy of Sciences, Pushchino, Russia

When synthesized inside a cell by an RNA polymerase, an RNA molecule must fold up into a particular structure that is required to mediate the molecule's biological activity. Complete knowledge of the folding properties of an RNA includes understanding both the structure of the final folded form and the process by which the folding occurs. Although many 3-dimensional RNA structures are being discovered, little is known about the mechanism of RNA folding. We focus on understanding the kinetics of RNA folding, including characterization of folding intermediates.

We are examining a large highly structured RNA, the self-splicing ribozyme from Tetrahymena thermophila. This RNA consists of 2 structural subdomains and has been characterized in great detail at the biochemical level. In addition, the crystal structure of one of the domains has been solved. Because of the wealth of information available on it, the ribozyme is an excellent model system for studies on the kinetics of folding. One of the structural domains folds rapidly, on the timescale of seconds. Only after this domain forms can the second structural domain form; formation of the second domain takes place on the timescale of minutes. Thus, the folding pathway is hierarchical, because 1 of the 2 domains must fold first, and the domains do not fold in parallel.

Folding of the Tetrahymena ribozyme, and large RNAs in general, is hampered by stalling in kinetic traps. The very forces that stabilize the final folded form of the RNA structure can also stabilize the folding intermediates, and thereby impede progress along the folding pathway. We discovered point mutations in the ribozyme that accelerate folding by destabilizing these kinetically trapped intermediates (Fig. 1). In addition, RNA folding can be accelerated by adding denaturants.

Formation of RNA tertiary structure usually requires the presence of divalent ions such as magnesium. Binding of magnesium ions stabilizes both secondary and tertiary structures of RNA, but too much magnesium can also overstabilize folding intermediates. Such overstabilization causes a slowing of folding. As was the case with the point mutations and denaturants, we found that ionic conditions that destabilize both the final folded form and the intermediate forms accelerate the rate of folding. RNA folding is slow at high concentrations of magnesium ions and fast at low concentrations of the ions. Thus, an optimal concentration of magnesium ions exists that balances the stabilization of RNA structure with rapid folding kinetics. Interestingly, for the Tetrahymena ribozyme, the optimal concentration is quite close to the concentration thought to be present under physiologic conditions inside cells.


Agalarov, S.C., Prasad, G.S., Funke, P.M., Stout, C.D., Williamson, J.R. Structure of the S15,S6,S18-rRNA complex: Assembly of the 30S ribosome central domain. Science 288:107, 2000.

Agalarov, S.C., Williamson, J.R. A hierarchy of RNA subdomains for protein binding in the assembly of the central domain of the 30S ribosomal subunit. RNA 6:402, 2000.

Ha, T., Zhuang, X., Kim, H.D., Orr, J.W., Williamson, J.R., Chu, S. Ligand-induced conformational changes observed in single RNA molecules. Proc. Natl. Acad. Sci. U. S. A. 96:9077, 1999.

Hennig, M., Williamson, J.R. Detection of N-H . . . N hydrogen bonding in RNA via scalar couplings in the absence of observable imino proton resonances. Nucleic Acids Res. 28:1585, 2000.

Mao, H., White, S.A., Williamson, J.R. A novel loop-loop recognition motif in the yeast ribosomal protein L30 autoregulatory RNA complex. Nat. Struct. Biol. 6:1139, 1999.

Mao, H., Williamson, J.R. Assignment of the L30-mRNA complex using selective isotopic labeling and RNA mutants. Nucleic Acids Res. 27:4059, 1999.

Mao, H., Williamson, J.R. Local folding coupled to RNA binding in the yeast ribosomal protein L30. J. Mol. Biol. 292:345, 1999.

Rook, M.S., Treiber, D.K., Williamson, J.R. An optimal Mg2+ concentration for kinetic folding of the Tetrahymena ribozyme. Proc. Natl. Acad. Sci. U. S. A. 96:12471, 1999.



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