Doctor of Philosophy, Case Western Reserve University, 2016, Biochemistry

The eukaryotic translation initiation factor 4F (eIF4F) consists of the large scaffolding and RNA binding protein eIF4G, the cap-binding protein eIF4E, and the DEAD-box RNA helicase eIF4A. In Saccharomyces cerevisiae, another DEAD-box RNA helicase, Ded1p, is also required for translation initiation. Here we show that eIF4A and Ded1p directly interact with each other and simultaneously with eIF4G. Both eIF4A and eIF4G interfere with the oligomerization of Ded1p. We delineate a comprehensive thermodynamic framework for the interactions between Ded1p, eIF4A, eIF4G, RNA and ATP, which indicates that eIF4A, with and without eIF4G, acts as modulator for activity and substrate preferences of Ded1p, which is the RNA remodeling unit in all complexes. We further show that Ded1p allows eIF4F to associate with short RNAs, which eIF4F alone cannot bind. Ded1p also dramatically enhances the binding of eIF4F to the 5'-cap. Our results reveal and characterize an unexpected interdependence between the two RNA helicases and eIF4G, and suggest that Ded1p is an integral part of eIF4F.

Committee: Eckhard Jankowsky (Advisor); William Merrick (Committee Chair); Coller Jeff (Committee Member); Derek Taylor (Committee Member); Blanton Tolbert (Committee Member) Subjects: Biochemistry

Master of Science in Biology, Cleveland State University, 2008, College of Science

Transcript-specific translational control restricts macrophage inflammatory gene expression. The pero-inflammatory cytokine IFN-γ induces the phosphorylation of human ribosomal protein L13a and its subsequent release from 60S ribosome. L13a is a component of the interferon-gamma-activated inhibitor of translation (GAIT). The GAIT complex binds a defined element in the 3'-untranslated region (UTR) of ceruloplasmin (Cp) mRNA and causes delayed silencing of translation. In this research, we elucidate the molecular mechanism underlying L13a translational silencing activity. L13a mediates translational silencing particularly, when driven by internal ribosome entry sites (IRESs) that requires the initiation factor eIF4G, but is resistant to silencing when driven by eIF4F- independent IRESs. This demonstrates a critical role of the scaffold protein eIF4G. Global inhibition of protein synthesis by targeting eIF4G is well appreciated in virus infection and apoptosis; however interaction of L13a with eIF4G blocks the 43S complex recruitment showing a unique role of eIF4G in gene specific translational silencing.

Committee: Barsanjit Mazumder PhD (Advisor); Anton Komar PhD (Committee Member); Crystal Weyman PhD (Committee Member) Subjects: Cellular Biology; Molecular Biology

Doctor of Philosophy, Case Western Reserve University, 2009, Biochemistry

DEAD-box proteins are involved in virtually all aspects of eukaryotic RNA metabolism. As members of the helicase superfamily 2 (SF2), DEAD-box proteins utilize ATP hydrolysis to unwind RNA, assemble large protein complexes on RNA, and remodel RNA protein complexes (RNPs). In the cell however, DEAD-box proteins function in the context of RNPs during processes such as splicing and translation. How the biochemical activities of DEAD-box proteins are utilized in a physiological setting is an important and central question in RNA metabolism. In this thesis, we address this issue by examining RNP remodeling and cofactor modulation by the DEAD-box protein Ded1p.We demonstrated that Ded1p did not actively displace the RNA binding proteins U1A and TRAP from their cognate RNA binding sites. Additionally, we established that the context of a RNP determined active displacement by Ded1p and propose a model for RNP remodeling by DEAD-box proteins. We found that that an inability to actively displace other proteins from RNA can provide non-sequence specific DEAD-box proteins with the capacity to disassemble similar RNA complexes in a discriminatory fashion. We further identified a physiologically relevant interaction between Ded1p and the translation initiation factor eIF4G. We showed that eIF4G did not modulate Ded1p ATPase activity, but did inhibit strand separation by Ded1p. Interestingly, Ded1p greatly increased eIF4G's affinity for RNA even to RNAs too small for eIF4G to bind alone. Our results suggest that Ded1p's biochemical activities facilitate eIF4G RNA binding. We propose a basic model for Ded1p enhancement of eIF4G RNA binding which may be relevant for Ded1p's role during translation initiation.

Committee: Eckhard Jankowsky (Advisor); Martin Snider (Committee Chair); Hung-Ying Kao (Committee Member); Pieter deHaseth (Committee Member) Subjects: Biochemistry; Molecular Biology