2025 Theses Doctoral

Investigating the contributions of eIF3 subunits to translation initiation

Ide, Nicholas Anthony

The transfer of genetic information is a necessity for all life on earth. The final step in this information flow is the translation of messenger RNA (mRNA) into protein by the ribosome. The molecular machinery responsible for translation includes the ribosome itself and a suite of factors responsible for getting the ribosome onto the mRNA at the correct time in a process called translation initiation. In addition to correct timing, translation initiation must also both be highly accurate and begin at the correct location (called the start codon), otherwise the incorrect protein product will be made.

This process involves several factors across all domains of life. In eukaryotes, these factors are called eukaryotic initiation factors (eIFs). There are over a dozen eIFs that are all required for the process of translation initiation. The primary focus of this thesis is on the molecular mechanisms used by one of these factors, a protein complex called eIF3. However, because eIF3 makes extensive functional and/or physical interactions with other eIFs and functions throughout many of the mechanistic steps of translation initiation, the insights gained into eIF3 function inform the understanding of the process of translation initiation broadly.

Generally, when studying molecular mechanism, it is often necessary and always desired to have the most control of the system under study as possible. In the case of eIF3, a large fraction of our knowledge about its function over the previous decades has come from in vivo genetic or cell-extract-based studies, which inherently lack precise control over components. While these studies have provided immense information regarding the impact of eIF3 across translation initiation and are the foundation for all future work, they lack information regarding the molecular mechanisms eIF3 employs.

Therefore, to develop a system capable of interrogating such mechanisms, Chapter 2 of this thesis presents a recombinantly reconstituted eIF3 complex that is functional in vitro and is used as a basis for site-specific fluorophore labeling of an individual eIF3 subunit. This biochemically active, reconstituted eIF3 is used throughout the rest of the thesis as it allows for enhanced control over the composition and identity of the eIF3 species under investigation.

In Chapter 3, the mRNA-binding activity of eIF3 is explored. The mRNA-binding activity ascribed to eIF3 is shown to be largely driven by a single eIF3 subunit, with the other subunits modulating this activity. Specifically, the largest eIF3 subunit, eIF3a, is shown to be the primary driver of mRNA-binding by eIF3 and the interactions of other subunits are shown to alter the observed binding. Following up on the study of mRNA-binding by individual eIF3 subunits, Chapter 4 expands to the study of eIF3 subunits and their coordination in additional biochemical activities, including the ability to bind to the ribosome itself. Lastly, the involvement of eIF3 in the attachment of mRNA to the ribosome is investigated. Specifically, Chapter 5 investigates the role of eIF3 in the process of loading the mRNA into the ribosome, a process that necessitates the handoff of the mRNA from the eIFs responsible for initial recognition of the mRNA to the ribosome.

Collectively, this thesis establishes a toolkit for studying the mechanistic roles of individual eIF3 subunits throughout translation initiation and uses this toolkit to describe how a single factor can accomplish such a wide range of biochemical activities.