Characterization of the editing activity and tRNA recognition in Escherichia coli leucine tRNA synthetase

Aminoacyl-tRNA synthetases are responsible for the aminoacylation of tRNA. This family of enzymes is essential to the fidelity of protein synthesis since they establish the genetic code by matching each amino acid with its cognate tRNA. Aminoacylation errors lead to genetic code ambiguity and incorrect proteins that are detrimental to cellular function. Some synthetases have editing activities that clear the wrong amino acid by hydrolysis of either of two substrates-misactivated aminoacyladenylates ("pre-transfer" of amino acid to tRNA) or misacylated tRNA ("post-transfer").;While post-transfer editing can be directly measured, pre-transfer editing is difficult to demonstrate, this is because adenylates are inherently labile and transient, and activity occurs against a background of post-transfer editing. Herein, different mutations in Escherichia coli leucyl-tRNA synthetase are combined to unmask the pre-transfer pathway. The mutant enzymes completely lack post-transfer editing, but prevent misacylations by clearing misactivated adenylates. We hypothesize that these mutations isolate a pre-transfer translocation step that moves misactivated adenylates from the activation site for editing. Mutant E. coli LeuRSs were also utilized to study the specificity of translocation for cognate versus non-cognate substrates. We suggest that LeuRS may utilize molecular signals to specifically translocate only the products that need to be hydrolyzed to prevent releasing misacylated tRNA.;The aaRSs must also recognize their specific tRNA out of a pool of structurally similar tRNAs. In order to block the misaminoacylation of an incorrect tRNA, aaRSs utilize molecular signals such as identity elements and antideterminants within tRNAs to confer or obstruct RNA-protein interactions. Interestingly Saccharomyces cerevisiae cytoplasmic LeuRS recognizes and aminoacylates E. coli tRNALeu, but E. coli LeuRS does not utilize S. cerevisiae tRNA Leu as a substrate.;Regions within the D-loop of S. cerevisiae tRNA Leu were mutated in order to confer cross-species aminoacylation of S. cerevisiae tRNALeu by E. coli LeuRS. Overlapping determinant patterns within the S. cerevisiae tRNALeu sequences were also observed for E. coli tRNATyr. Similar mutations were generated to allow misaminoacylation of E. coli tRNATyr with leucine by E. coli LeuRS. We hypothesize that the identity elements of tRNA Tyr and tRNALeu in E. coli have co-evolved to prevent misaminoacylation.