Defects in Transient tRNA Translocation Bypass tRNA Synthetase Quality Control Mechanisms

Quality control mechanisms during protein synthesis are essential to fidelity and cell survival. Leucyl-tRNA synthetase (LeuRS) misactivates non-leucine amino acids including isoleucine, methionine, and norvaline. To prevent translational errors, mischarged tRNA products are translocated 30Å from the canonical aminoacylation core to a hydrolytic editing-active site within a completely separate domain. Because it is transient, the tRNA translocation mechanism has been difficult to isolate. We have identified a “translocation peptide” within Escherichia coli LeuRS. Mutations in the translocation peptide cause tRNA to selectively bypass the editing-active site, resulting in mischarging that is lethal to the cell. This bypass mechanism also rescues aminoacylation of an editing site mutation that hydrolyzes correctly charged Leu-tRNA^Leu. Thus, these LeuRS mutants charge tRNA^Leu but fail to translocate these products to the hydrolytic site, where they are cleared to guard against genetic code ambiguities.Quality control during translation depends on the family of aminoacyl-tRNA synthetases (aaRSs),² which is responsible for the first step of protein synthesis. Each aaRS selectively aminoacylates just one of the 20 standard amino acids to its cognate tRNA (1). About half of this family of enzymes ensures fidelity by employing a “double sieve model” that relies on two active sites (2, 3). One sieve is synthetic and produces charged tRNA. The other is a hydrolytic editing-active site that clears mistakes. Defects in the editing mechanism cause cell death (4, 5) and also neurological disease in mammals (6).The aminoacylation site in the ancient canonical core of the aaRS activates its cognate amino acid but can also misactivate structurally similar amino acids (1). The editing-active site blocks the correctly charged amino acid (7, 8) and hydrolyzes mischarged amino acids from the tRNA. Amino acid editing destroys mistakes before they can be incorporated by the ribosome, which would result in the production of statistical proteins (1).Amino acid proofreading requires that the charged tRNA transiently migrates between two enzyme domains that are responsible for aminoacylation and editing. For leucyl-tRNA synthetase (LeuRS) and the homologous isoleucyl-(IleRS) and valyl-tRNA synthetases (ValRS), the editing domain resides in a structural insertion called CP1 (9) that splits the Rossmann ATP binding fold. The insert folds independent of the canonical core (10–12). The isolated CP1 domains from LeuRS, ValRS, and IleRS can independently and specifically hydrolyze mischarged amino acid from its cognate tRNA (13–15).The aminoacylation and editing-active sites of LeuRS are separated by about 30 Å. Thus, the charged 3′ end of the tRNA must be faithfully translocated a significant distance for proofreading and then hydrolysis if it is mischarged (16). It has also been suggested that the tRNA 3′ end binds initially near the editing-active site and requires translocation to the aminoacylation site (17).We hypothesized that flexible molecular hinges might facilitate conformational changes between the aminoacylation and the editing complexes (18). Two putative hinge sites were predicted by computational analysis of Thermus thermophilus LeuRS. One hinge at Ser-227 was located in the N-terminal β-strand that links the aminoacylation and CP1 editing domains (18). Mutations at the predicted hinge site in the β-strand linker of Escherichia coli LeuRS abolished aminoacylation activity and significantly decreased amino acid editing activity (18).A second hinge site at Glu-393 was identified in a flexible peptide within the CP1 domain of T. thermophilus LeuRS (18). Here, we describe results at a homologous Asp-391 site in E. coli LeuRS that demonstrate that this hinge comprises a portion of a translocation peptide. Unlike the predicted β-strand hinge mutation, the aminoacylation and editing activities of the CP1 domain-based hinge mutants in LeuRS were intact. Surprisingly however, mutations within the translocation peptide yield mischarged tRNA despite a robust deacylation activity. We hypothesize that impairing the LeuRS translocation peptide causes the charged tRNA 3′ end to bypass the editing sieve prior to product release. Defects in the translocation peptide and its mechanism result in amino acid toxicities that are lethal to the cell.