The proposed study is designed to combine our ongoing studies on acetylcholinesterase and nicotinic acetylcholine receptors in order to examine structure-function relationships and gene expression in the cholinergic nervous system. The approach relies heavily on recombinant DNA methods, the structure of genes encoding these two proteins (some of which we have cloned), the development of a library of mutant and chimeric proteins, monitoring expression of genes in cells in culture and in situ, and physical methods to examine protein structure. Studies with acetylcholinesterase structure-function are based on a high resolution crystal structure and can be carried to atomic level resolution. Our interests are directed to entry routes of ligands, the influence of particular amino acid side chains on orientation and environment of bound ligands, dissection of the acylation and deacylation steps, and communication between the active center gorge and the bulk solvent. The acetylcholine receptor structure is only known to 8-10 angstroms resolution, and one faces the prospect of another decade or two elapsing before having a high resolution structure. Nevertheless, valuable information on structure-function can be garnered from a mutagenesis approach. Our interests here encompass the identification of residues at ligand recognition sites, the involvement of subunit interfaces in ligand specificity and subunit association, and the state transitions that produce cooperativity. We have found that the enhanced acetylcholinesterase mRNA associated with muscle cell differentiation is associated with m NA stabilization while nicotinic receptor mRNA levels are controlled by transcription. We plan to examine mRNA stabilization in detail and extend these considerations to neurons and the hematopoietic system. To this end, we propose to delineate the elements in mRNA structure important for mRNA stabilization and the signalling mechanisms linking cell differentiation to mRNA and transcriptional control of gene expression.
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