40S ribosomal subunits move along the 5' UTR through an eIF4A-independent mechanism in higher eukaryotes.

During translation initiation, the 40S small ribosomal subunit is recruited to the mRNA 5' cap and scans the 5' untranslated region (UTR) to locate the start codon. While the mechanism of 40S translocation remains elusive, the RNA helicase eIF4A has long been suspected as the primary molecular motor driving 40S scanning. In this study, we utilized GFP reporter mRNAs to investigate the impact of 5' UTR length on translational efficiency. We found that an 8-fold variation in the length of unstructured 5' UTRs did not lead to substantial changes in translation efficiency in wheat germ extract (WGE) and human HEK293T cell lysate. By contrast, the presence of a stable stem-loop in the middle of the 5' UTR significantly reduced cap-dependent translation. These results suggest that mRNA scanning is not rate-limiting when the UTR is devoid of secondary structure. Inhibition of eIF4A by hippuristanol in cell-free protein synthesis systems yielded an equivalent decrease in translation for mRNAs with short and long unstructured 5' UTRs, indicating that eIF4A may be dispensable for 40S scanning. Our data suggest that helicase-independent one-dimensional diffusion may be the primary mechanism enabling 40S movement along the 5' UTR during initiation.