News and Publications

RNA Folding

* 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 folding.

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.

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. RNA 4:984, 1998.

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.