Isolated CP1 Domain of Escherichia coli Leucyl-tRNA Synthetase Is Dependent on Flanking Hinge Motifs for Amino Acid Editing Activity

Abstract

Protein synthesis and its fidelity rely upon the aminoacyl-tRNA synthetases. Leucyl-(LeuRS), isoleucyl-(IleRS) and valyl-(ValRS) tRNA synthetases have evolved a discrete editing domain called CP1 that hydrolyzes the respective incorrectly misaminoacylated noncognate amino acids. Although active CP1 domain fragments have been isolated for IleRS and ValRS, previous reports suggested that the LeuRS CP1 domain required idiosyncratic adaptations to confer editing activity independent of the full-length enzyme. Herein, characterization of a series of rationally designed E. coli LeuRS fragments showed that the β-strands, which link the CP1 domain to the aminoacylation core of LeuRS are required for editing of mischarged tRNA Leu . Hydrolytic activity was also enhanced by inclusion of short flexible peptides that have been called "hinges" at the end of both LeuRS β-strands. We propose that these long β-strand extensions of the LeuRS CP1 domain interact specifically with the tRNA for post-transfer editing of misaminoacylated amino acids.The fidelity of protein synthesis relies upon the interpretation of genetic information by the aminoacyl-tRNA synthetases (aaRSs) (1,2). Each of the aaRSs is responsible for aminoacylation of its cognate amino acid to a specific set of tRNA isoacceptor molecules. In † This work was supported by the National Institutes of Health (Grant GM63789) and The Robert A. Welch Foundation (E-1404) To whom correspondence should be addressed: Department of Biochemistry, University of Illinois at Urbana-Champaign, Roger Adams Laboratory, Box B4, 600 South Mathews Avenue, Urbana, IL 61801-3732. Phone: 217-244-2405. Fax: 217-244-5858 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript a two-step reaction, the enzyme binds ATP and its cognate amino acid to form an activated aminoacyl-adenylate intermediate. In the second step, the amino acid moiety is transferred to the 3′ end of the tRNA.Amino acids that are similar in size, shape, and/or polarity threaten enzyme specificity (3-6). For example, discrimination of amino acids that differ by one methyl group such as isoleucine and valine, was theoretically predicted to yield an error rate that was as high as 1 out of 5 (4). However, a number of aaRSs have evolved amino acid editing mechanisms to clear these types of mistakes (6). Separate active sites for aminoacylation and editing are employed in a 'double sieve' model to enhance amino acid selection and discrimination (7,8). The first 'coarse' sieve contains the aminoacylation active site, which activates cognate amino acids along with structurally similar noncognate amino acids. The second 'fine' sieve is an editing active site, which hydrolyzes noncognate amino acids that are mischarged, but importantly, excludes the correctly charged amino acids.Leucyl-(LeuRS), isoleucyl-(IleRS) and valyl-(ValRS) tRNA synthetases are homologous enzymes that belong to subclass IA of the aaRSs (9). These enzymes hydrolyze mischarged amino acids using a discrete domain called connective p...

Coordination of tRNA Synthetase Active Sites for Chemical Fidelity

“…The Nand C-terminal ␤-strands of the CP1 domains were fused to the Rossmann fold of M. mobile LeuRS at Glu-229 and Gly-232 (⅐⅐⅐ 224 WIGKEEIDG 232 ⅐⅐⅐), respectively. This corresponds to Glu-228 and Ala-362, the naturally occurring E. coli LeuRS fusion sites that link the flexible ␤-strands of the CP1 domain to the Rossmann fold (19,25).…”

In vitro assays for the determination of aminoacyl-tRNA synthetase editing activity

“…The first coarse sieve, an aminoacylation active site that is located in an ancient canonical core of the aaRS, is responsible for activating amino acids. A second fine sieve, a hydrolytic active site, has been clearly demonstrated to correct the enzyme's mistakes in a number of synthetase systems (27)(28)(29)(30)(31)(32)(33)(34)(35).…”

Yeast Mitochondrial Leucyl-tRNA Synthetase CP1 Domain Has Functionally Diverged to Accommodate RNA Splicing at Expense of Hydrolytic Editing

“…1A) (20). Although the LeuRS CP1 domain is best known for its role in proofreading or editing misacylated tRNA Leu (21,22), genetic rescue experiments identified splicing sensitive sites within and around the CP1 domain (9,23). We had also shown that the isolated CP1 domain from ymLeuRS stimulated bI4 intron RNA splicing in vivo (17).…”

“…1B) extends from Ile-260 to Glu-431. Previously we showed that the ␤-strands are required for deacylation of mischarged tRNA in the E. coli enzyme (21). Thus, we constructed a CP1-containing fragment that included the ␤-strands, extending from Trp-238 to Leu-446 (CP1-␤; Fig.…”

“…1B). We had also determined that inclusion of short extensions from the ␤-strands to the aminoacylation core that contained conserved WIG and RDW motifs enhanced tRNA deacylation by the isolated E. coli CP1 domain (21). In addition to the ␤-strands, these extensions have been shown to be important for tRNA Leu -protein interactions in E. coli LeuRS (21, 30, 31).…”