Many ways in which large RNA molecules and RNA-protein complexes participate in gene expression are now known, and the structures of these RNA are of considerable interest. The secondary structures of several large RNAs are well-understood, and attempts are being made to determine their overall three dimensional folding. As one approach to this problem, we notice that a number of small structural units are conserved among different RNAs. These structural motifs can be reproduced in small, synthetic RNAs amenable to detailed physical studies. Measurement of bend, twist, and hydrogen bonding of a motif could be incorporated into a three dimensional model of a large RNA. Unusual properties of a motif, such as an increased flexibility or alternate conformations, might provide a functional rationale for its conservation. The RNA structures we intend to study are helical segments, with both canonical and non-canonical base pairs; bulges; purine-rich internal loops; and pseudoknots. Several different techniques will be used to determine the structures of these motifs: Gel electrophoresis methods developed in this lab can measure the twist associated with a helix-like structure; Electric birefringence measurements of rotational diffusion can accurately measure end-to-end length of RNAs, determining rise per base pair and bend angles; Non-radiative energy transfer measurements can estimate the hinge-like flexibility of a bulge or loop structure; Closed circular RNAs can be used to estimate the torsional stiffness of bulge or loop structures; Melting experiments can measure the stabilities of unusual structures.
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