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Despite the similarity in fundamental goals of translation initiation between different domains of life, it is one of the most phylogenetically diverse steps of the central dogma of molecular biology. In a classical view, the translation signals for prokaryotes and eukaryotes are distinct from each other. This idea was challenged by the finding that the Internal Ribosome Entry Site (IRES) belonging to Plautia stali intestine virus (PSIV) could bypass the domain-specific boundaries and effectively initiate translation in E. coli. This finding led us to investigate whether the ability of PSIV IRES to initiate translation in E. coli is specific to this IRES and also to study features that allow this viral IRES to mediate prokaryotic translation initiation. We observed that certain IRESs may also possess the ability to initiate E. coli translation. Our results also indicated that the structural integrity of the PSIV IRES in translation in prokaryotes does not appear to be as critical as it is in eukaryotes. We also demonstrated that two regions of the PSIV IRES with complementarity to 16S ribosomal RNA are important for the ability of this IRES to initiate translation in E. coli.