Initiation of eukaryotic protein synthesis begins with separated 40S and 60S ribosomal subunits. The first step is formation of a 48S initiation complex at the initiation codon of mRNA. This process requires ternary complex, elFs 3, 1, 1A, 4A, 4B, 4F and a 40S subunit. The 40S subunit in 48S initiation complexes is associated with initiation factors and therefore can not immediately bind a 60S subunit. Joining of a 60S subunit to a 48S complex is associated with two linked events: hydrolysis of GTP bound to elF2 in the 48S complex and displacement of factors. Hydrolysis of elF2-bound GTP is stimulated by elF5. Recently we showed that hydrolysis of elF2-bound GTP is not sufficient to promote ribosomal joining as had previously been proposed, and that a second novel factor termed eIF5B is required. eIF5B is a homolog of the prokaryotic initiation factor IF2 and has a ribosome-dependent GTPase activity that is essential for its function. We shall identify and characterize structural elements of eIF5B responsible for the interaction of this factor with other translation components (40S and 60S subunits, Met-tRNAi, initiation factors) and the role of such interactions in subunit joining. Experiments will be done to determine whether eIF5 and eIF5B simply prepare 48S complexes for subsequent subunit joining or actively participate in the process. The roles of eIF5 and eIF5B in the displacement of initiation factors and the mechanism of displacement will also be determined. We will investigate the mechanism of ribosomal stimulation of eIF5B's GTPase activity and particular the role of ribosomal P proteins in this process. The localization of eIF5B on the ribosome will be studied by directed hydroxyl radical probing, chemical and enzymatic foot-printing and cryo-electron microscopy. These data will provide the basis for understanding the role of elF5B in subunit joining, and the mechanism of stimulation of eIF5B's GTPase activity by the ribosome. Fast kinetics techniques (fluorescence stopped-flow and quench-flow) will be applied to resolve individual steps and to study kinetic mechanism of two hydrolysis steps in subunit joining: the hydrolysis of elF2-bound GTP induced by elF5 and the hydrolysis of another GTP by elF5B.
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