This proposal is designed to elucidate the mechanisms that regulate translation of the mRNAs encoding transition proteins, protamines and the mitochondrial capsule seleno-protein in haploid spermatogenic cells in mice. The central aim is to determine whether the observation that protamine 1 messenger ribonucleoprotein complexes (mRNPs) form initiation complexes with 80s single ribosomes slower than deproteinized protamine 1 mRNAs in brief incubations in the reticulocyte cell free lysate reflects the mechanisms that repress translation of protamine 1 mRNA in vivo. The kinetics of formation of initiation complexes in vitro will be compared for mouse protamine 1 mRNAs in deproteinized poly(A) plus mRNA, translationally repressed mRNPs, translationally active mRNPs and translationally repressed mRNPs after disruption of mRNP complexes with high salt. The interactions of cis-acting sequences in protamine 1 mRNAs and trans-acting mRNP proteins will be analyzed by synthesizing radiolabeled run off protamine 1 transcripts with bacteriophage RNA polymerases, constituting mRNP particles by incubation with cytoplasmic extracts, and translation in cell free system and gel mobility shift assays. Non-isotopic in situ hybridization will be used to determine whether protamine 1 mRNAs are sequestered intracellularly in round spermatids. This proposal is intended to introduce promising undergraduates to research in molecular biology.
