Translation initiation is one of the key regulatory steps in the protein biosynthesis pathway. Prior research identified numerous variations in this mechanism optimizing cellular performance during specific conditions. Of particular interest is an alternative mode of translation initiation via an internal ribosomal entry site (IRES), that is associated with stress response. A multitude of IRES-specific trans-acting factors (ITAFs) have been identified. In this investigation, we hypothesized that many ITAFs remain undiscovered. We conducted a large-scale screen in S. cerevisiae using a LacZ
reporter under IRES control to identify genes positively regulating IRES-mediated initiation. Six genes were selected as potential ITAFs, and their genetic interaction networks were explored for the links to the translation process. We found associations between our candidates and the genes involved in both translation and stress response mechanisms, supporting their proposed function as ITAFs and demonstrating the potential for further investigations to identify regulators of IRES-mediated translation.