The Conformational Dynamics of eIF5B During Ribosomal Subunit Joining

ABSTRACT Translation initiation in eukaryotes, which requires over a dozen eukaryotic initiation factors (eIFs), is a

highly regulated process that helps establish the fidelity of protein synthesis and is directly targeted during cellular stress and disease. To add to the complexity of this process, ribosomal initiation complexes (ICs) undergo multiple conformational rearrangements upon eIF binding and activity, and dysregulation of the associated

conformational changes are thought to underlie cellular stress and disease. Despite the importance of these molecular rearrangements for the function of the translation machinery, current structural approaches cannot resolve the real-time dynamics of the eIF binding events and IC structural rearrangements that are inferred from

comparisons of the static structural snapshots provided by X-ray crystallographic and cryogenic electron microscopy studies. This presents a gap in our understanding of how eIF binding and eIF- and ribosome structural rearrangements cooperatively ensure the fidelity of translation initiation. Specifically, the last step in

the initiation pathway, in which the large ribosomal subunit joins to the IC, functions as a final regulatory checkpoint in the pathway. This step is regulated by the translational guanosine triphosphatase (GTPase) eIF 5B and eIF 1A. Specifically, 5B accelerates the subunit joining step in a GTP- and methionylated initiator tRNA

(Met-tRNAi)-dependent manner that is facilitated by 1A. This leads to questions such as: Do dynamic structural rearrangements of 5B underlie its ability to accelerate subunit joining? If so, how do GTP and Met-tRNAi modulate the dynamics of 5B? Likewise, how does 1A modulate the dynamics of 5B and how does such

modulation regulate the ability of 5B to recognize Met-tRNAi, accelerate subunit joining, and undergo GTP hydrolysis upon subunit joining? To address these questions, I will use a combination of advanced single- molecule Förster Resonance Energy Transfer (smFRET) techniques and ensemble biochemical and biophysical

approaches to characterize the dynamic structural rearrangements of 5B, 1A, and the IC during subunit joining, and elucidate the contributions that these dynamics make to the mechanism and regulation of translation initiation. The immediate goal of this proposal is to dissect the conformational dynamics and kinetics of 5B and

1A, demonstrating how these eIFs couple Met-tRNAi recognition to acceleration of subunit joining, GTP hydrolysis by 5B, and dissociation of 5B and 1A following productive subunit joining.