Biology subjects

Aitken, C. E.

Publications and source records attributed to Aitken, C. E..

4 recordsLinked to original sources

bioRxiv2026

Dynamic redistribution of eIF4F controls cap-dependent translation initiation

Translation initiation requires messenger RNAs (mRNAs) to be recognized and loaded into ribosomes through a process catalyzed by the heterotrimeric eukaryotic initiation factor eIF4F. During this process, eIF4F engages the 7-methylguanosine cap at the 5 end of the mRNA and promotes productive engagement with the ribosomal pre-initiation complex (PIC) to facilitate PIC loading onto the mRNA. Although eIF4F is central to translation initiation and its regulation, the molecular mechanism by which eIF4F stimulates PIC loading, and the mechanistic role of the essential ATP hydrolysis step catalyzed by eIF4F, have remained unresolved. Here, we use single-molecule fluorescence microscopy to directly visualize the dynamics of eIF4F during cap recognition and PIC engagement. We show that ATP binding, but not ATP hydrolysis, promotes productive assembly of eIF4F on mRNA and enables dynamic redistribution of eIF4F along the transcript. In contrast, ATP hydrolysis is specifically required for recycling of cap-stalled eIF4F during productive PIC engagement. Furthermore, we identify eIF3 and eIF4B as the minimal PIC-associated factors required to stimulate ATP-hydrolysis-dependent recycling of eIF4F during PIC loading. Together, our results support a model in which productive PIC engagement stimulates ATP-hydrolysis-dependent recycling of eIF4F, thereby coupling eIF4F recycling to PIC loading during translation initiation. This mechanism provides a framework for understanding how mRNA topology, RNA-binding proteins, and the availability of initiation factors can control translational efficiency.

biophysics↗

bioRxiv2024

A dynamic compositional equilibrium governs mRNA recognition by eIF3

Eukaryotic translation initiation factor (eIF) 3 is a multi-subunit protein complex that binds both ribosomes and messenger RNAs (mRNAs) to drive a diverse set of mechanistic steps during translation of an mRNA into the protein it encodes. And yet, a unifying framework explaining how eIF3 performs these numerous activities is lacking. Using single-molecule light scattering microscopy, we demonstrate that Saccharomyces cerevisiae eIF3 is in dynamic exchange between the full complex, subcomplexes, and subunits. By extending our microscopy approach to an in vitro reconstituted eIF3 and complementing it with biochemical assays, we define the subspecies comprising this dynamic compositional equilibrium and show that mRNA binding by eIF3 is not driven by the full complex but instead by the eIF3a subunit within eIF3a-containing subcomplexes. Our findings provide a mechanistic model for the role of eIF3 in mRNA recruitment and establish a mechanistic framework for explaining and investigating the other activities of eIF3.

biophysics↗

bioRxiv2021

Cellular translational enhancer elements that recruit eukaryotic initiation factor 3

Translation initiation is a highly regulated process which broadly affects eukaryotic gene expression. Eukaryotic initiation factor 3 (eIF3) is a central player in canonical and alternative pathways for ribosome recruitment. Here we have investigated how direct binding of eIF3 contributes to the large and regulated differences in protein output conferred by different 5'-untranslated regions (5'-UTRs) of cellular mRNAs. Using an unbiased high-throughput approach to determine the affinity of budding yeast eIF3 for native 5'-UTRs from 4,252 genes, we demonstrate that eIF3 binds specifically to a subset of 5'-UTRs that contain a short unstructured binding motif, AMAYAA. eIF3 binding mRNAs have higher ribosome density in growing cells and are preferentially translated under certain stress conditions, supporting the functional relevance of this interaction. Our results reveal a new class of translational enhancer and suggest a mechanism by which changes in core initiation factor activity enact mRNA-specific translation programs.

systems biology↗

bioRxiv2017

eIF4A is stimulated by the pre-initiation complex and enhances recruitment of mRNAs regardless of structural complexity

eIF4A is a DEAD-box RNA-dependent ATPase thought to unwind RNA secondary structure in the 5'-untranslated regions (UTRs) of mRNAs to promote their recruitment to the eukaryotic translation pre-initiation complex (PIC). We show that eIF4As ATPase activity is markedly stimulated in the presence of the PIC, independently of eIF4E*eIF4G, but dependent on subunits i and g of the heteromeric eIF3 complex. Surprisingly, eIF4A accelerated the rate of recruitment of all mRNAs tested, regardless of their degree of structural complexity. Structures in the 5'-UTR and 3' of the start codon synergistically inhibit mRNA recruitment in a manner relieved by eIF4A, indicating that the factor does not act solely to melt hairpins in 5'-UTRs. Our findings that eIF4A functionally interacts with the PIC and plays important roles beyond unwinding 5-UTR structure is consistent with a recent proposal that eIF4A modulates the conformation of the 40S ribosomal subunit to promote mRNA recruitment.

biophysics↗