Extending the spacing between the Shine-Dalgarno sequence and P-site codon reduces the rate of mRNA translocation

By forming basepairing interactions with the 3’ end of 16S rRNA, mRNA Shine-Dalgarno (SD) sequences positioned upstream of Open Reading Frames (ORFs) facilitate translation initiation. During the elongation phase of protein synthesis, intragenic SD-like sequences stimulate ribosome frameshifting and may also slow down ribosome movement along mRNA. Here, we show that the length of the spacer between the SD sequence and P-site codon strongly affects the rate of ribosome translocation. Increasing the spacer length beyond six nucleotides destabilizes mRNA-ribosome interactions and results in a 5-10 fold reduction of the translocation rate. These observations suggest that during translation, the spacer between the SD sequence and P-site codon undergoes structural rearrangements, which slow down mRNA translocation and promote mRNA frameshifting.