The broad goal of the project is to elucidate the molecular mechanisms of initiation and control of protein synthesis in mammalian cells. Ten initiation factors comprising 25 unique polypeptides have been purified and characterized. cDNAs encoding three of these have been cloned in this lab and additional cDNA clones are being sought in order to obtain primary protein structures. The binding of initiation factors and ribosomes to mRNAs is being studied by using in vitro SP6 transcripts which can be readily labeled, biotinylated and/or altered in sequence. Correlations of initiation factor phosphorylation states and translational activity are being established by detecting phosphorylated factor forms through immunoblotting of cell lysate proteins fractionated on high resolution 2-dimensional gels. Attempts are being made to detect differences in the in vitro activities of phosphorylated and non-phosphorylated elF4B and elF4F. The multiple sites of elF4B phosphorylation and the cellular protein kinase(s) involved will be determined and a link to protein kinases associated with the plasma membrane will be sought: In addition to the in vitro studies of translational control, recombinant DNA techniques are being employed to develop approaches to studying protein synthesis in vivo. A mutant form of elF2 alpha that lacks the phosphorylation site is synthesized efficiently in transfected cells and appears to inhibit the dsRNA- regulated elF-2 alpha kinase. To continue this promising line of research, wildtype and mutant initiation factor cDNAs will be expressed transiently or in long-term transfected cells, with the aim of obtaining in vivo evidence that phosphorylation actually causes a change in the efficiency of initiation. Coordinate regulation of initiation factor gene expression will be studied with cDNA probes. Functional domains for the activity and assembly of elF2 will be studied both in vivo and in vitro by purification of overproduced subunits from cells transfected with wildtype or mutant cDNAs. The cellular levels of individual initiation factors will by increased or decreased by expression of their cDNAs or antisense RNAs in transfected cells, and effects on rates of protein synthesis monitored. Finally, the translation of the highly efficient elF2 alpha mRNA and the uncapped poliovirus mRNA will by probed to ascertain how each is recognized by the translational machinery. Thus, by a combination of in vitro tudies with purified components and in vivo approaches exploiting recombinant DNA techniques, detailed mechanisms of initiation factor activity and translational control will be elucidated.
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