The broad objective of this project is to elucidate the mechanism by which protein synthesis is regulated in the sea urchin egg. This area has been the subject of intensive study, both because of it's intrinsic importance as a key developmental event and also because it represents a model system for understanding a type of translational regulation which probably occurs in most cell types. The major thrust of this project will be two fold. It will attempt to determine the regulatory pathways by which the known ionic changes at fertilization signal the protein synthetic machinery to activate and to determine the actual components of the protein synthetic machinery which are acted upon to increase the rate of protein synthesis (i.e. masked mRNA, ribosomes, initiation factors, etc.). There will be extensive use of cell free translation systems in which the effects of signals such as changes in pH and calcium may be studied at the level of discrete steps in the process of initiation and elongation. Various parameters of translation will be compared in cell free systems prepared from both unfertilized and fertilized eggs to identify changes at levels other than ionic controls. Both an mRNA dependent nuclease treated translation system and a partially fractionated translation system will be developed to allow a more refined analysis of the protein synthetic machinery. The role of masking of mRNAs by protein repressors will be evaluated both in vivo and in vitro, using a series of novel assays. We will test the hypothesis that the increase in histone synthesis after fertilization is regulated by ionic controls both by direct measurements with ion specific microelectrodes and by manipulation of ionic conditions both in vivo and in vitro. In addition the effects of the calcium activated changes in arginine phophate and inorganic phosphate will be determined on the specific steps in protein synthesis. This part of the project will also use a novel method for measuring adenylate charge in a single intracellular compartment. The calcium regulated activation of protein synthesis may represent the first regulatory pathway in which we can with confidence identify the initial intracellular signal, the pathway by which the signal is mediated and the mechanism by which the protein synthetic machinery responds to that signal.
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