A novel mechanism of 5’ UTR structure in promoting prokaryotic translational initiation

Gene expression involves consecutive and delicate regulation of transcription and translation. One of the mechanisms that controls the initiation of translation is the structure of the 5’ untranslated region (5’ UTR) of mRNA. In prokaryotes, the initiation process depends on the ribosome binding site (RBS), which is a complementary sequence to the ribosomal RNA and provides an anchor of ribosome assembly. Current research has demonstrated that releasing the RBS from an aptamer or hairpin structure could resume translation. However, how structures upstream of the RBS 5’ UTR are involved in the process of translational control is poorly understood. Here we applied a cell-free in vitro translation system to investigate additional upstream 5’ UTR structures such as hairpins and RNA G-quadruplexes (rG4) located far from RBS. First, we discovered that rG4 strongly promotes translation efficiency compared to non-structural RNA, and the enhancement increased with addition of a hairpin structure. After systematically evaluating the rG4 structures, we found a strong correlation (r = 0.88) between translation efficiency and the bulkiness of rG4. We also elucidated that the increase in translation is unlikely to be caused by providing additional RBS or altering the accessibility of RBS. Finally, we demonstrated that the formation of structure is essential to enhance the translation. Consequently, our results support a novel mechanism where structures in the 5’ UTR can serve as a physical blockade to force ribosome to move forward instead of falling off from 5’ site of mRNA.