REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS INITIATION

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Initiation of protein synthesis in eukaryotic organisms is accomplished through one of the most complex series of biochemical reaction known. Knowledge of this process is important for an understanding of how the rate of protein synthesis is regulated, the cytotoxic effect of many viruses, the action of the antiviral protein interferon, and the ultimate expression of genetic information. The long-term objectives of this project are two- fold: to understand the biochemical roles of various proteins (elF- 4 group initiation factors) involved in the entry of messenger RNA into the initiation process, and to initiation factors responsible for the activity and level of expression of the initiation factor eIF-4E (cap-binding protein). For the first objective, the active site of eIF-4E will be determined by a combination two techniques: a set of new photoaffinity labels will be synthesized and used to label the m7GTP-binding site of eIF-4E. Second, altered forms of eIF-4E will be produced in vitro by site-directed mutagenesis. The location of eIF-4A, -4B, -4E and -4F on various initiation complexes will be determined. The interaction of cap structures, both in mRNA and in photoaffinity derivatives of m7GTP, with the eIF-4 group factors will be investigated. Finally, the cDNA for the p220 component of eIF-4F will be cloned and sequenced. For the second objective, the effect of phosphorylation of eIF-4E will be examined. This will be studied in vitro through the use of specific kinase and by cell-free synthesis of forms of eIF-4E lacking a phosphorylation site, produced by site-directed mutagenesis. It will also be studied in vivo, by correlating phosphorylation with protein synthesis rates and by using transient expression vectors containing mutagenized forms of eIF-4E cDNA. Finally, the structure of the various forms of eIF-4E mRNA will be examined, and the gene for this protein will be cloned and partially sequenced. R0GM33804 Selected functional properties that are characteristic of cytochrome c will be investigated and the possible endowment of this protein with new functional properties will be explored through the construction of a series of specifically designed mutants as follows: (1) The putative crystallographic identification of a substrate binding site on the surface of the Ser-82 variant will be evaluated by examination of the effect of relevant small molecules on the oxidation-reduction properties of the protein. (2) The role of the axial ligands in determining cytochrome function will be studied by analysis of Met-80 mutants. (3) The mechanism by which mutations at positions 38 and 82 alter the alkaline transition will be studied by pH-jump experiments, EPR spectroscopy, and electrostatics calculations. (4) The Takano Dickerson model for cytochrome c redox interconversion will be evaluated by study of recently constructed Thr-78 mutants. This residue is critical to the Takano-Dickerson model as it hydrogen- bonds to a crucial, internally-bound water molecule that is pivotal to their proposal. (5) The role of heme propionate-7 in regulating the reduction potential of the protein will be studied by consideration of a Tyr-48 mutant. Possible analysis of Tyr-48/Arg- 38 double mutants will be considered in this regard as well. (6) Further analysis of the multiple roles of Phe-82 will be analyzed by evaluation of several new mutants constructed at this position. (7) The origin of species differences between cytochromes will be considered through construction and characterization of loop insertion/deletion mutants which convert yeast iso-l cytochrome c into forms closer in size to two prokaryotic cytochromes. (8) The effects of selected mutations on the kinetics of electron transfer to physiological redox partner proteins will be studied. (9) A battery of spectroscopic techniques (NMR, CD/MCD, and time-resolved fluorescence spectroscopy) will be applied to selected mutants as dictated by their observed properties.