Large RNA molecules (e.g. ribosomal and messenger RNAs, RNA viruses, viroids) are capable of a variety of interactions and profoundly influence the expression of genetic information in a cell. The folding of these RNA molecules (secondary and tertiary structure) is important for their function, yet the structure of large RNA molecules has been a difficult problem to approach experimentally. Little is known about the mechanisms by which proteins interact with large RNA molecules and recognize specific sites. The long term aim of the proposed studies is to remedy this gap in our knowledge and understand general features of large RNA structures and interactions with proteins. The work proposed is a detailed study of RNA structure and protein interactions in two regions of the E. coli 16S ribosomal RNA; both regions are more complex than any RNA studied in detail to data. The simpler of the two regions, the S8/15 binding region, covers about 200 nucleotides in the middle of the RNA sequence and contains simple hairpin secondary structures and one long range interaction; two proteins specifically bind in this region. The S4 protein recognition region encompasses the 5 feet half of the molecule and includes extensive secondary interactions between distant parts of the RNA sequence. S4 also binds a site on its own mRNA; the structure of this recognition site will also be studied for comparison with the ribosomal binding site. New methods and strategies for manipulating large RNA molecules have already been developed in this lab and will be applied to these systems. These include a procedure for excising precise RNA fragments from large molecules; the fragment are suitable for 'footprinting' experiments using chemical reagents to probe RNA structure and protein interactions. A method for attaching fluorescent reporter groups sensitive to RNA secondary structure at specific sites within large RNA molecules will be used to look the thermodynamics of RNA folding. By combining chemical modification experiments to look at the thermodynamics of interactions, a complete picture of RNA folding and protein binding in these two 16S RNA regions should be obtained.
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