PHYSICAL STUDIES OF RNA STRUCTURAL MOTIFS

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Many different RNA molecules are involved in gene expression in cells. In the last few years the secondary structures of several important large RNAs have been deducedd by sequence comparison methods, and it is becoming clear that certain structural motifs are important for the function of these RNAs. The object of this proposal is to study the three dimensional structures and thermodynamics of formation of these structural motifs. Synthetic RNAs will be designed to reproduce these structures in molecules small enough to be amenable to detailed physical studies. The specific motifs to be studied are single base bulges (important for protein recognition); pseudoknots, or base pairing between hairpin loops (probably involved in structural rearrangements of RNAs); and helices containing large proportions of non-canonical base pairs. Using in vitro transcription of cloned synthetic DNA by purified T7 polymerase, 20-30 base RNA molecules containing these structures will be synthesized. 10 milligram amounts can be prepared when necessary. Several kinds ouf physical measurements will be made to determine the structure and thermodynamics of these RNAs: melting temperatures of related RNAs, binding constants for intercalator - RNA or RNA - RNA interctions, and proton NMR spectra. In addition, chemical modification studies of these model RNAs will provide a way to compare their conformations with those of large RNAs.