Initiation Factors in Eukaryotic Protein Biosynthesis

Maitra, Umadas

Abstract

The P.I.'s laboratory is specifically involved in studying mammalian translation initiation particularly with regard to the interaction of translation initiation factors with ribosomes, initiator Met-tRNAf, GTP, and mRNA during 40S and 80S initiation complex formation. Both biochemical studies with purified mammalian initiation factors as well as molecular genetic studies using the yeast Saccharomyces cerevisiae are to be used to understand the function and regulation of activity of several key translation initiation factors in vitro and in vivo. In the current grant proposal, the following areas will be pursued. a) Translation initiation factor eIF5, that interacts with the 40S initiation complex (40S eIF3 AUG Met-tRNAf eIF2 GTP)and mediates the hydrolysis of bound GTP,will be characterized as follows. (i) The significance of the specific interaction of eIF5 with the beta subunit of eIF2 in promoting GTP hydrolysis will be investigated by generating mutant eIF5 proteins that do not interact with eIF2 beta and then assaying the ability of these mutant proteins for eIF5 activity in vitro. These mutant proteins will also be tested for their ability to maintain yeast cell growth and viability when present in yeast cells as the only source of eIF5. (ii) Interaction of eIF5 with known or novel components of the translation initiation apparatus that modulate the GTPase-promoting activity of eIF5 on the 40S preinitiation and 40S initiation complexes will be studied. (iii) The significance of phosphorylation of eIF5 in the regulation of eIF5 activity in vivo will be analyzed. (b) An efficient in vitro translation initiation system that requires initiation factors eIF1A and eIF3 for the transfer of the Met-tRNAf eIF2 GTP ternary complex to 40S ribosomal subunits in the absence of mRNA has been developed. Using such a performed 40S initiation complex (40S eIF3 Met-tRNAf eIF2 GTP) as the substrate, the requirements for mRNA binding to form the 40S initiation complex and its subsequent interaction with eIF5 and 60S subunits to form the 80S initiation complex will be studied. (c) The role of initiation factor eIF6 in 80S initiation complex formation will be investigated. This 26- kDa protein binds to 60S ribosomal subunits and prevents the association between 40S and 60S ribosomal subunits. However, the resulting 60S eIF6 complex is unable to participate in the formation of the 80 initiation complex, indicating that a mechanism must exist for the release of eIF6 from the 60S ribosomal subunit prior to 80S initiation complex formation. The factor(s) involved in the release of eIF6 from the 60S eIF6 complex will be identified and characterized.