40S ribosomal subunits scan mRNA for the start codon by one-dimensional diffusion

During eukaryotic translation initiation, the small (40S) ribosomal subunit is recruited to the 5ʹ cap and subsequently scans the 5ʹ untranslated region (5ʹ UTR) of mRNA in search of the start codon. The molecular mechanism of mRNA scanning remains unclear, particularly the requirement for and identity of a translocase. Here, using GFP reporters in Saccharomyces cerevisiae, we show that order-of-magnitude variations in the length of unstructured 5ʹ UTRs have only modest effects on protein synthesis, while structured 5’ UTRs strongly inhibit translation. Thus, when not hindered by secondary structure, mRNA scanning is not rate limiting. Loss-of-function mutations in eIF4A, Ded1 and Slh1 reveal that these translational helicases are dispensable for mRNA scanning. Our data suggest that one-dimensional diffusion predominately enables 40S movement along the 5ʹ UTR during mRNA scanning.