A G-quadruplex (G4) structure in the 5' untranslated region (5'UTR) of mRNA enhances translation in a size-dependent manner. This study used a T7-based in vitro translation system and E. coli to investigate how G4 formation at the 5'UTR affects translation. The results show that G4 strongly promotes translation efficiency, with a 12-fold increase when a hairpin is inserted upstream. The effect is not due to increased ribosome affinity, ribosome binding site accessibility, or mRNA stability. Instead, a physical barrier model is proposed, where bulky structures in the 5'UTR bias ribosome movement toward the downstream start codon, increasing translation output. The study provides insights into the regulatory role of 5'UTR structures in translation, highlighting their potential applications in tuning gene expression. The effect of G4 on translation is size-dependent, with longer loops leading to higher translation efficiency. The presence of a hairpin upstream further enhances translation. The study also shows that the effect of G4 is not due to increased ribosome binding or mRNA stability, but rather a physical barrier that biases ribosome movement. The findings were validated in both in vitro and E. coli systems, demonstrating that the 5'UTR structure-dependent translational enhancement exists in both systems. The study suggests that the bulkiness of the upstream structure is more essential than the type of structure. The results indicate that the G4 structure acts as a physical barrier that biases ribosome movement toward the translation start site. The study also shows that the effect of G4 on translation is not due to increased ribosome binding or mRNA stability, but rather a physical barrier that biases ribosome movement. The findings were validated in both in vitro and E. coli systems, demonstrating that the 5'UTR structure-dependent translational enhancement exists in both systems. The study suggests that the bulkiness of the upstream structure is more essential than the type of structure. The results indicate that the G4 structure acts as a physical barrier that biases ribosome movement toward the translation start site.A G-quadruplex (G4) structure in the 5' untranslated region (5'UTR) of mRNA enhances translation in a size-dependent manner. This study used a T7-based in vitro translation system and E. coli to investigate how G4 formation at the 5'UTR affects translation. The results show that G4 strongly promotes translation efficiency, with a 12-fold increase when a hairpin is inserted upstream. The effect is not due to increased ribosome affinity, ribosome binding site accessibility, or mRNA stability. Instead, a physical barrier model is proposed, where bulky structures in the 5'UTR bias ribosome movement toward the downstream start codon, increasing translation output. The study provides insights into the regulatory role of 5'UTR structures in translation, highlighting their potential applications in tuning gene expression. The effect of G4 on translation is size-dependent, with longer loops leading to higher translation efficiency. The presence of a hairpin upstream further enhances translation. The study also shows that the effect of G4 is not due to increased ribosome binding or mRNA stability, but rather a physical barrier that biases ribosome movement. The findings were validated in both in vitro and E. coli systems, demonstrating that the 5'UTR structure-dependent translational enhancement exists in both systems. The study suggests that the bulkiness of the upstream structure is more essential than the type of structure. The results indicate that the G4 structure acts as a physical barrier that biases ribosome movement toward the translation start site. The study also shows that the effect of G4 on translation is not due to increased ribosome binding or mRNA stability, but rather a physical barrier that biases ribosome movement. The findings were validated in both in vitro and E. coli systems, demonstrating that the 5'UTR structure-dependent translational enhancement exists in both systems. The study suggests that the bulkiness of the upstream structure is more essential than the type of structure. The results indicate that the G4 structure acts as a physical barrier that biases ribosome movement toward the translation start site.