mRNA's in eukaryotic cells are translated either on free or on membrane-bound ribosomes. The events which lead to the exclusive translation of certain mRNA's on membrane-bound ribosomes and the subsequent proteolytic processing of the nascent chains synthesized on membrane-bound polysomes will be investigated. Cells or tissues using membrane-bound polysomes largely for the synthesis of secretory proteins (myeloma, pancreas) or membrane proteins (brain) or both (liver) will be fractionated. Rough microsomes or derived bound polysomes will be separated from free polysomes. In vitro readout of their mRNA's or isolation of mRNA's from these fractions followed by translation in heterologous systems (Krebs Ascites, rabbit reticulocytes, wheat germ) will be used to obtain a nonprocessed, primary translation product. The mechanism of subsequent proteolytic modification of the primary in vitro translation product will be studied using peptide fingerprinting and sequencing techniques. In vitro reconstitution of rough microsomes from isolated components under conditions of protein synthesis will be attempted; mRNA's derived from free polysomes or from bound polysomes will be used to discriminate between nonspecific aggregation and bona fide reconstitution. The sites of two tightly bound distinct proteins on the nontranslated regions of a large variety of mRNA's will be investigated. Fingerprinting of the sites may reveal nucleotide sequences (other than the already described polyadenylate) common to all mRNA's.
NIH ExPorter
NIH ExPorter