News and Publications

RNA Folding

* 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.

Publications

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. 26:1991, 1998.

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, 1998.

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.