Abstract:

Transfer RNAs (tRNAs) adopt a stable L-shaped tertiary structure crucial for their involvement in protein translation. Among various divalent metal ions, magnesium ions play a pivotal role in preserving the tertiary structure of tRNA. However, the precise location of the Mg²⁺ binding pocket in human tRNA remains elusive. In this investigation, we identified the Mg²⁺ binding site within human tRNA^Gln using suppressor tRNA^Gln. This variant of tRNA recognizes premature stop codons (specifically UAG) and facilitates the expression of full-length proteins. By mutating sites U8 and C72 in suppressor tRNA^Gln, we assessed the decoding efficiency of the resulting mutant suppressor tRNAs, which serves as a measure of tRNA’s ability to decode genetic information. Our analysis revealed that the U8C mutant suppressor tRNA exhibited a significantly lower Mg²⁺ content compared to the C72U mutant. Furthermore, we observed a notable reduction in decoding efficiency in the U8-mutated suppressor tRNA, as evidenced by GFP fluorescence and Western blotting analysis. Conversely, mutations at the C72 site had a comparatively minor impact on decoding efficiency. These findings underscored the tight binding of Mg²⁺ to the U8 site of human tRNA^Gln, crucial for maintaining the stability of tRNA tertiary structure and translation efficacy. Additionally, our investigation delved into the influence of glutamine availability on tRNA decoding efficiency at the cellular level. The results indicated that both the concentration of amino acids and the codon context of TAG could modulate tRNA decoding efficiency. This study provided valuable insights into the structure and function of tRNA, laying the groundwork for further exploration in this field.

Key words: Metal ions, tRNA tertiary structure, Glutamine supply, Decoding efficacy