PHYSICAL AND GENETIC STUDIES OF RNA STRUCTURES

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The aim of this proposal is to study in detail the structure and dynamics of two regions in the E. coli 16S ribosomal RNA, recognizing either S4 protein (greater than 500 bases) or S8 and S15 proteins (approximately 200 bases). The objective of the work is to understand the principles governing the folding and interaction of large RNA molecules. Three complementary approaches will be used: Medium resolution mapping of RNA structures: specific RNA fragments covering the regions of interest will be prepared and probed with structure specific reagents to delineate the RNA secondary structures, regions of major tertiary interactions, and points of contact with bound proteins. The protein binding affinity of fragments will be compared to intact 16S rRNA. Detailed physical studies: once a fairly small fragment of RNA (less than 150 bases) is found to have particularly interesting structural or protein binding features, it will be prepared in quantity for studies by optical and NMR spectroscopy, and its crystallization for x-ray diffraction studies will be attempted. These physical methods are capable of giving a very high resolution picture of the folding, interactions, and dynamics of a limited RNA molecule. The first studies will be with a 112 base fragment containing the S15 binding site and known to have unusually high affinity sites for intercalators. Genetic studies: General schemes will be devised for selecting mutations deficient in a protein - RNA recognition; these will be carried out with S4 protein and the ribosomal and messenger RNAs able to bind it specifically. Mapping of the mutation sites will define the essential RNA and protein features for binding and direct the physical studies to important regions.