The experiments in this proposal are designed to explore the mechanism of initiation of translation in cells from higher eukaryotes. The objectives are to identify structural features in mRNA that direct ribosomes to the correct AUG codon for initiation, to elucidate structural features that modulate the efficiency of translation, and to try to understand how those features function. Past experiments, in which the expression of a cloned preproinsulin gene was studied in transfected COS cells, identified a patch of nucleotides around the AUG codon (GCCACCAUGG) that affects the efficiency of initiation over a 10- to 20-fold range. Attempts are underway to reproduce the effects context in vitro, using wheat germ and reticulocyte translation systems. Other experiments are proposed to: (1) Analyze SP64-CAT transcripts that have a strong hairpin structure (delta G -63 kcal/mol) upstream from the AUG initiator codon. The main prediction to be tested is whether the 40S ribosomal subunit binds to the mRNA, scans part way and then stalls on the 5' side of the hairpin. (2) Investigate the possibility of long-distance secondary structure effects on translation; (3) Extend the study of an SV40-insulin construct called B13hp, in which a hairpin with a predicted stability of -30 kcal/mol inhibits translation in vivo only under conditions of hypertonic stress. The translation of B13hp will be studied under other conditions of stress and in cells that have been synchronized with respect to the cell cycle. Other experiments are designed to look for the induction of an RNA- unwinding activity in extracts from cells that have become conditioned to high salt, and to determine (using oligonucleotide probes) whether the predicted hairpin in constructs like B13hp actually exists under the conditions of translation. (4) The ability of R17-phage coat protein to inhibit in vitro translation of an SP64-CAT transcript will be studied as a model system to learn how eukaryotic ribosomes respond to obstruction by proteins. (5) In vitro experiments will be designed to probe the mechanism by which context facilitates recognition of the AUG codon and the mechanism by which a long 5' noncoding sequence enhances translation. If ribosomes can be induced to reinitiate translation in vitro, the requirements for reinitiation will be studied.
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