Structural and molecular basis of specialized translation mediated by the ribosome mRNA-binding channel

The ribosome mRNA channel is central to translation, yet its role in regulatory mechanisms remains unclear. Using cryo-EM of human ribosomal complexes bound to Kozak and TISU mRNAs from wild-type (WT) and RPS26/eS26 mutant (RPS26dC) cells, we demonstrate that both RPS26/eS26 and mRNA adopt distinct conformations, explaining the opposing effects of RPS26dC on their activity. Translatome studies of WT and RPS26dC reveal AUG-context-dependent changes in 48S and 80S initiation complexes and slower scanning. Downregulated mRNAs are enriched for specific AUG-upstream nucleotides and a -1-cytosine contacting 18S rRNA G1207, an interaction lost in RPS26dC. Strongly affected transcripts include replication-dependent histones, which, despite short 5'UTRs and suboptimal Kozak, exhibit robust translation activity that is RPS26/eS26-dependent. We identify a translational enhancer in the H2B 5'UTR (-16 to -9) overlapping predicted RPS26/eS26-binding sites, with a distinct ribosome-bound conformation. Exploiting these features, we engineered a high-efficiency translational cassette with minimal leaky scanning. These findings underscore the role of the ribosome's mRNA channel in selective translation and its therapeutic potential.