Sortase-Mediated Fluorescent Labeling of eIF4E for Investigating Translation Initiation Mechanisms

Translation initiation represents a critical regulatory step in gene expression, orchestrated by the interaction of eukaryotic initiation factor 4E (eIF4E) with the 7-methylguanosine (m⁷G) cap structure at the 5' end of mRNA. This interaction enables eIF4F, composed of eIF4E, eIF4G, and eIF4A, to recruit the 43S preinitiation complex to the mRNA 5' end. The activity of eIF4E is tightly regulated and often dysregulated in cancer, neurological disorders, and viral infections. To investigate the interactions of human eIF4E with m⁷G-RNA and eIF4G, we engineered single-cysteine mutants of eIF4E to enable fluorescent dye attachment. However, these mutants presented challenges in purification and exhibited diminished activity. To overcome these issues, we developed a method to fluorescently label eIF4E via sortase-mediated transpeptidation. Our results demonstrate that sortase-labeled eIF4E retains activity comparable to wild-type eIF4E. This approach provides a valuable tool for studying the dynamic mechanisms of translation initiation and its regulation.