News and Publications
The Skaggs Institute for Chemical Biology
Scientific Report 1999-2000
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
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.