John William Baker Hershey - US grants

DESCRIPTION (provided by the applicant): The overall goal of the project is to elucidate the detailed molecular mechanisms whereby translational controls contribute to the regulation of gene expression in eukaryotic cells. A broad experimental approach is taken that involves biochemical, biophysical, recombinant DNA and genetic techniques to study the structure, function and regulation of specific initiation factors in human and yeast cells. Two general hypotheses will be tested: i) That elF3 plays an essential organizational/structural role in forming multi-initiation factor complexes on the 40S ribosomal subunit; and ii) That the phosphorylation of elF3 and eIF4B enhances the activities of these factors and stimulates initiation of protein synthesis. The principal investigator will exploit the recent finding that essentially all of the individual subunits of human eIF3 can be expressed as stable tagged or untagged proteins in baculovirus-infected insect cells but not in bacteria, and that an active 5-subunit core complex can be formed. He proposes to generate and purify various sub-complexes of eIF3 for functional and high-resolution structural studies. The core subunits that bind to elF1, 4B, 4G and 5 and to the ancillary eIF3 subunits will be identified. The role of the p33 subunit in the function of yeast eIF3 will be elucidated by a mutant suppressor screen and by determining its physiologic RNA binding target by the SELEX procedure. Investigation of eIF3 subunit mRNA levels by kinetic RT-PCR, protein levels by Western immunoblotting and mRNA translation efficiencies by polysome profile analyses will determine which subunit genes are coordinately regulated or which may be overexpressed, thereby possibly serving other functions. The principal investigator's prior work shows that both eIF3 and eIF4B phosphorylation correlates with translation activation. Work to identify phosphorylation sites and responsible kinases will continue for eIF4B, and be extended to the 4 phosphorylated eIF3 subunits, especially p110 which interacts with many other initiation factors. Effects of phosphorylation will be tested for individual subunits by expression of wild type and site-substituted mutant forms in the baculovirus system, followed by measurement of their interaction with other proteins. The experiments will help elucidate the function and regulation of these key initiation factors.

Protein synthesis is an integral part of gene expression, and translational control mechanisms are known to operate during the initiation phase of the process. The projects proposed here are designed to elucidate the molecular events of initiation, with special emphasis on the synthesis and mechanism of action of the three initiation factors IF1, IF2, and IF3. First, the expression of initiation factor genes in Escherichia coli will be studied by recombinant DNA techniques. The genes for IF1 (infA), IF2 (infB) and IF3 (infC) already have been cloned, mapped and sequenced. We propose to test whether or not IF1 is required for cell viability, by inactivating infA on the bacterial chromosome. Operon and protein fusion of infA and lac will be used to determine whether or not infA expression is regulated by IF1 levels (autogenous regulation) or by the stringent response. The coordinate regulation of all three initiation factor genes as a function of growth rate also will be studied in vivo will lac fusions. Overproducing strains for initiation factors will be constructed and isolation methods will be developed that employ fast protein liquid chromatography to purify the proteins. Second, we propose to study in vivo the physiological consequences of reducing the cellular levels of each of the initiation factors. In initial studies with IF2, infB was placed under control of the lac promoter/operator, and expression was regulated by the concentration of the inducer, IPTG. Strains with infA and infC under lac promoter control will be constructed and we shall study the effects of reduced factor levels on protein synthesis rates, polysome profiles, rRNA operon expression, specific proteins synthesized, and initiation pathway intermediates. Third, the mechanism of action of initiation factors will be probed by in vitro mutagenesis and by fluorescence polarization techniques. Both random and site-directed mutagenesis will be employed and the resulting mutated genes will be screened in vivo by transformation of the genes into strains carrying the chromosomal gene under lac promoter control. Growth characteristics in the absence of IPTG will depend on the mutated factor. Interesting mutant forms will be overexpressed and the mutated factor purified and studied by a variety of in vitro assays for function. Continuation of fluorescence polarization studies of factor- ribosome interactions will address quantitatively the binding of individual initiation factors to 30S and 70S initiation complexes in order to shed light on the reaction pathway of initiation. The studies should result in an increase in our knowledge of the initiation process, which is essential for understanding mechanisms of protein synthesis and translational control at the molecular level.

Regulation of mRNA translation constitutes an important aspect in the control of gene expression mammalian cells. Most examples of translational control involve modulation of the initiation phase of protein synthesis. Two kinds of initiation events can be distinguished either of which may be regulated: 1) initiation on polysomes, thereby maintaining a full complement of ribosomes on the mRNAs; and 2) initiation on messenger ribonucleoprotein (mRNP) particles, thereby mobilizing non- active mRNAs into active polysomes. The latter event is poorly understand, yet its regulation is encountered frequently. The PIs propose to characterize proteins that bind to mRNP particles and that mask their activiies. One such protein of 50 kDa has been purified in Pushchino, and its mechanism of action as a translational inhibitor will be probed. Its site of binding to mRNAs will be determined, and the inhibited step of protein synthesis will be identified. They also plan to clone its cDNA and determine the effect of overexpression in transfected mammalian cells. Preliminary evidence from Puschino suggests that the 50 kDa protein may itself be regulated by an "activator"; the "activator" will be purified and characterized. These studies rely on classical biochemical techniques of protein purification and chemistry presently used in both Davis and Puschino, employ in vitro cell-free translatiion systems, some highly fractionated and reconstituted with purified initiatiion factors from Davis, and employ state-of-the-art transfection methods. %%% The project is designed to shed light on the basic molecular mechanisms of how mRNP particles are activated and inhibited for translation, and thus should help elucidate this major poorly understood area of gene regulation.

The major goal of the project is to understand how the human translation initiation factor elF3 contributes to the initiation mechanism and to the regulation of viral mRNAs, especially Hepatitis C Virus (HCV) mRNA that initiates translation by ribosome binding to an internal entry site (IRES). The structure of elF3, a factor comprising 12 non-identical subunits, will be probed by isolating a variety of sub-complexes formed in baculovirus-infected insect cells. The binding of elF3 and its sub-complexes to the HCV IRES will be defined quantitatively. A highly active partially fractionated mammalian cell lysate system dependent on purified elF3 will be constructed with (5-globin mRNA. This system will be used to define the role of elF3 and its subcomplexes in the translation of HCV mRNA. An in vitro translation system also will be constructed that utilizes all of the purified initiation factors, in order to define which factors are required for the translation of HCV and Dengue virus mRNAs. These studies will determine to what extent the viral mRNAs differ from capped/polyadenylated mRNAs in their initiation mechanisms, and may identify targets for therapeutic intervention of infections by these viruses. Considerable evidence indicates that elF3 plays an important role in regulating protein synthesis and cell proliferation. elF3, like other initiation factors, is phosphorylated on numerous subunit proteins. The sites of phosphorylation will be identified by mass spectroscopy, and the effects of phosphorylation on function will be tested in the translation assays. Mutant forms will be constructed and tested, with phosphorylation sites substituted with alanine to prevent phosphorylation, and with glutamate or aspartate to mimic phosphorylation. The experiments are designed to elucidate the mechanism of action of elF3 and the role of phosphorylation in regulating its activity during initiation of capped and IRES-driven mRNAs. The results may explain how disfunction or disregulation of elF3 may affect the control of cell proliferation, resulting in celt malignancy.