News and Publications
The Skaggs Institute For Chemical Biology
Scientific Report 1997-1998
J.R. Williamson, S. Agalarov,* I. Baxter, R. Burris, C.D. Cilley, K.T. Dayie,
P. Funke, H. Mao, J.W. Orr, P.K. Radha, M.S. Rook, L.G. Scott, D.K. Treiber,
* Russian Academy of Sciences, Pushchino, Russia
** Bryn Mawr College, Bryn Mawr, PA
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 biological
activity of the molecule. 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 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 1 domain 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 folding kinetics. The first is a kinetic oligonucleotide
hybridization assay. In this assay, we monitor the time-dependent accessibility
of different regions of the RNA to binding by an oligonucleotide probe after
initiating folding by adding magnesium ions. In the second assay, we monitor
the gain of catalytic activity of the ribozyme after addition of magnesium as
the folding proceeds. Using these 2 tools, we have 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 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 (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 accelerate formation of the second domain are 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 have 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 are the facts that addition of denaturants accelerates
folding and that the mutations lessen the effect of urea.
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 the 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. Nucleic
Acids Res., in press.
Brodsky, A.S., Erlacher, H.A., Williamson, J.R. NMR evidence for a
base triple in the HIV-2 CGC-mutant TAR argininamide complex. Nucleic Acids Res.
Brodsky, A.S., Williamson, J.R. Solution structure of the HIV-2 TAR-argininamide
complex. J. Mol. Biol. 267:624, 1997.
Dayie, K.T., Tolbert, T.J., Williamson, J.R. 3D C(CC)H TOCSY experiment
of assigning protons and carbons in uniformly 13C- and selectively 2H-labeled
RNA. J. Magn. Reson. 130:97, 1997.
Orr, J.W., Hagerman, P.J., Williamson, J.R. Protein and Mg2+-induced
conformational changes in the S15 binding site of 16S ribosomal RNA. J. Mol.
Biol. 275:453, 1998.
Tolbert, T.J., Williamson, J.R. Preparation of specifically deuterated
and 13C-labeled RNA for NMR studies using enzymatic synthesis J. Am.
Chem. Soc. 119:12100, 1997.
Treiber, D.K., Rook, M.S., Zarrinkar, P.P., Williamson, J.R. Kinetic
intermediates trapped by native interactions in RNA folding. Science 279:1943,
Zamore, P.D., Williamson, J.R., Lehmann, R. The Pumilio protein binds
RNA through a conserved domain that defines a new class of RNA-binding proteins.
RNA 3:1421, 1997.