New Study Reveals the Yin and Yang of tRNA

Transfer RNA (tRNA) plays the central role in the catalytic processes of LeuRS, including aminoacylation, tRNA-dependent pre- and post-transfer editing reactions. All of these functions of LeuRS rely on the intra-molecular translocation of tRNA CCA-tail between the synthetic and editing domains. Now a new study from Chinese Academy of Sciences reveals that the proper binding of acceptor end determines the catalytic balance of aminoacylation and editing.

The free tRNA CCA-tail preferentially binds to the CP1 domain and exhibits a regular helical conformation favoring binding to the discrete editing site under the condition that no amino acid is activated. In the aminoacylation conformation, the 3’ end of the tRNA CCA-tail distorts to bring the 2’-hydroxyl of the terminal ribose into the required position for attacking the aminoacyl-adenylate intermediate, suggesting that cis-elements of LeuRS are required to accomplish the translocation from the low energy state to the high energy state. Dr. TAN Min, Mr. WANG Meng and their colleagues under the guidance of Professor WANG Enduo showed that R418 of EcLeuRS was an essential element assisting the translocation of tRNA CCA-tail from the editing domain to synthetic domain. Substitution of R418 with negative-charged Glu or Asp would repel with tRNA during its intra-molecular translocation, resulting in almost abolished aminoacylation activity. Previous crystal structure study showed that E292 of the CP1 domain and R416 of the synthetic domain form a salt-bridge in the aminoacylation conformation. The present work revealed the function of this salt bridge, maintaining the uncharged tRNA in the synthetic site before aminoacylation reaction. Either E292R or R416E single mutation broke the salt-bridge due to electrical charge repel. As a consequence, tRNA could prefer to translocate to its low energy state with tRNA CCA-tail binding in the editing domain, leading to severely impacted aminoacylation activity. Combination of E292R and R416E restored the salt-bridge and recovered the aminoacylation activity to a level comparable to that of the WT enzyme.

Furthermore, disturbance of tRNA intra-molecular translocation with EcLeuRS also affected the editing activity of the enzyme. The greater binding of tRNA CCA-tail in the editing domain, which was a consequence of the entry of tRNA into synthetic domain being blocked, would significantly enhance tRNA-dependent pre-transfer editing. These results, together with previous work by Prof. WANG’s group, provide a comprehensive assessment of how intra-molecular translocation of the tRNA CCA-tail balances the aminoacylation and editing activities of LeuRS. In addition, this work raises the possibility of new antibiotic development targeting the intra-molecular translocation of tRNA within aminoacyl-tRNA synthetases.

This study entitled “The Yin and Yang of tRNA: proper binding of acceptor end determines the catalytic balance of editing and aminoacylation” was published online in the Nucleic Acids Research on April 12^th, 2013. It is funded by grants from the Ministry of Science and Technology of China, the National Natural Science Foundation of China, Chinese Academy of Sciences, and the Science and Technology Commission of Shanghai Municipality.

The intra-molecular translocation of tRNA CCA-tail within LeuRS balances its aminoacylation and editing activities. (Image by Prof. WANG Enduo)