Browsing by Subject "Escherichia coli"

Transfer RNA (tRNA) is one of the most extensively modified RNA molecules. The role of tRNA modifications become apparent during physiological condition such as oxidative stress, where it serves as an adaptive response to the changing environment. These modifications are upregulated mainly at the wobble position of the tRNA to enhance the translational efficiency of the stress response genes through enhanced decoding rate and tRNA–mRNA interaction. Hence, tRNA modification has a crucial role in regulating translational fidelity, and such modifications can be utilized to fine-tune the translation for improved production of heterologous protein. Therefore, this study aimed to analyze the tRNA modification changes in two laboratory-significant E. coli strains (BL21 (DE3) and K12) during oxidative stress and utilize these modifications to enhance the production of heterologous protein using a defined cell-free protein synthesis system. Ultra-performance liquid chromatography-mass spectrometry was used to detect and quantify the tRNA modification changes in the hydrogen peroxide-treated E. coli cells. The results showed unique tRNA modification patterns and intensities between the two bacterial strains in response to oxidative stress. Modifications such as ac4c and m2,2G were upregulated in E. coli BL21 (DE3) following hydrogen peroxide treatment, whereas k2C and chm5U were increased in E. coli K12. Further analysis of the dataset revealed that most of the upregulated ribonucleoside modifications were predominant at the anticodon loop of the tRNAs, indicating the potentiality of these tRNA pools to impact on translation. Likewise, I optimized an E. coli-based cell-free protein synthesis system to investigate the effect of modified tRNA pools on translation. Hence, this study serves as a stepping stone to understand the tRNA modification landscape of E. coli and provides a platform to depict the function of post-transcriptional tRNA modifications in translation with the CFPS system.