News and Publications
The Skaggs Institute for Chemical Biology
Scientific Report 1998-1999
J.R. Williamson, D. Abramovitz, S. Agalarov,* I. Baxter, R. Burris, C.D. Cilley,
K.T. Dayie, V. Feher, P. Funke, M. Hennig, H. Mao, J.W. Orr, P.K. Radha, 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 its biological
activity. Complete knowledge of the folding properties of an RNA requires 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 the nature of folding intermediates.
Most large RNAs contain considerable amounts of secondary structures that
form extremely rapidly. These secondary structures are typically held together
by weaker tertiary interactions that usually require stabilization by binding
of divalent ions such as magnesium. Initially, we are probing a later part of
the folding of RNA, the cascade of events that occurs after addition of magnesium
ions. We hope to learn about the rates of folding and the nature of the intermediates
along the folding pathway.
We are focusing on 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 knowledge available on this RNA, the ribozyme is an excellent model system
for studies on the kinetics of folding.
We use 2 assays to monitor the folding kinetics. The first is a kinetic oligonucleotide
hybridization assay. With this assay, we monitor the time-dependent accessibility
of different regions of the RNA to binding by an oligonucleotide probe after
we initiate folding by adding magnesium ions. With the second assay, we monitor
the gain of catalytic activity of the ribozyme after addition of magnesium as
the folding proceeds. Using these tools, we developed a basic picture of the
events along the folding pathway. One of the 2 structural domains folds rapidly,
on a timescale of seconds. Only after this domain forms can the second structural
domain form; formation of the second domain takes place on a timescale of minutes.
Thus, the folding pathway is hierarchical, because 1 of the 2 domains must fold
first, and the domains to not fold in parallel (Fig. 1).
The folding rate for the 2 domains differs greatly, and we were interested
in the kinetic barrier that makes formation of the second domain relatively slow.
To understand this barrier, we devised an in vitro selection strategy to detect
mutations that accelerate folding. The results were quite surprising. All the
mutations that accelerated formation of the second domain were located in the
first domain. We had expected that the second domain folds slowly because it
is misfolded and that we would find mutations that disrupt the misfolded form.
Instead, we found mutations that destabilized the native structure already formed
in the first domain. On the basis of these results, we proposed that a kinetic
trap is formed because the first domain is extremely stable, so stable that it
restricts conformational searching necessary for formation of the second domain.
Consistent with this idea is the fact that addition of denaturants accelerates
Currently, we are characterizing other folding intermediates in the same way.
A picture of RNA folding is emerging in which the RNA must escape from a series
of kinetic traps to reach the native state. Apparently, the forces that make
RNA so stable can also impede formation of the final structure.
Batey, R.T., Williamson, J.R. Effects of polyvalent cations on the
folding of an rRNA three-way junction and binding of ribosomal protein S15. RNA
Rook, M.S., Treiber, D.K., Williamson, J.R. Fast folding mutants of
the Tetrahymena group I ribozyme reveal a rugged folding energy landscape.
J. Mol. Biol. 281:609, 1998.
Zamore, P.D., Bartel, D., Lehmann, R., Williamson, J.R. The PUMILIO-RNA
interaction: A single RNA-binding domain monomer recognizes a bipartite target
sequence. Biochemistry 38:596, 1999.