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.