The Interactions Between Escherichia Coli Leucyl-tRNA Synthetase And TRNA～(Leu) In Aminoacylation And Editing

Escherichia coli leucyl-tRNA synthetase (LeuRS) has a large connecting polypeptide (CP1) inserted into its active site. It was demonstrated that the peptide bond between E292-A293 was crucial for the aminoacylation activity of E. coli LeuRS. To investigate the effect of E292 on the function of E. coli LeuRS, E292 was mutated to K, F, S, D, Q and A. These mutations at 292 did not change the specific activity of the amino acid activation reaction. Though the conformational change of these mutants was not detected in CD spectroscopy and fluorescence emission spectroscopy, their aminoacylation activities were impaired to varying extents. The mutation of E to K decreased the aminoacylation activity to the largest extent. Analysis of the Km values of these mutants for the three substrates showed that the E292 was not involved in the binding of leucine and that all mutants had stronger binding with ATP.However, these LeuRS mutants can mischarge tRNALeu isoacceptors, indicating that the editing ability of these LeuRS mutants were impaired to varying extents also. For the first time, we found that the LeuRS mutants can discriminate two tRNALeu isoacceptors, tRNALeu 1or tRNALeu 2. The structural basis for this is that the first base pair of tRNALeu acceptor arm is Watson-Crick base pair or wobblebase pair. The results indicate that the flexibility of the first base pair can affect the editing reaction catalyzed by LeuRS. It seems that the flexibility of the first base pair of tRNALeu may probably affect the mischarged 3'-end of tRNALeu shuttling from synthetic site to editing site, and that transferred acceptor arm of tRNALeu may interact with LeuRS in the region around E292.To ensure the fidelity of protein biosynthesis, aminoacyl-tRNA synthetases (aaRSs) must recognize the tRNA identity elements of their cognate tRNAs and discriminate their cognate amino acids from structure similar ones through a proofreading (editing) reaction. For a better understanding of these processes, we systematically investigated the roles of tertiary base pairs at "elbow region" of tRNALeu in the aminoacylation and editing reactions catalyzed by LeuRS, found that any disturbance of the tertiary interaction between the tRNALeu D- and T?C-loops affected both its aminoacylation ability and its ability to stimulate the editing reaction. 19:56 played important roles in these two reactions. Moreover, both were closely correlated and dependent on the hydrogen bond number. In contrast, U54:A58 was more important in aminoacylation reaction than in editing reaction. Our results suggest that elbow region of tRNA formed by the tertiary interactions between the D- and T?C-loops affect the interactions between LeuRS and tRNALeu effectively both in aminoacylation and in editing.