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Attenuation of the Editing Activity of the Escherichia coli Leucyl-tRNA Synthetase Allows Incorporation of Novel Amino Acids into Proteins in Vivo

Tang, Yi and Tirrell, David A. (2002) Attenuation of the Editing Activity of the Escherichia coli Leucyl-tRNA Synthetase Allows Incorporation of Novel Amino Acids into Proteins in Vivo. Biochemistry, 41 (34). pp. 10635-10645. ISSN 0006-2960. doi:10.1021/bi026130x.

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The fidelity of translation is dependent on the specificity of the aminoacyl-tRNA synthetases (aaRSs). The aaRSs that activate the hydrophobic amino acids leucine, isoleucine, and valine employ a proofreading mechanism that hydrolyzes noncognate aminoacyl adenylates and misaminoacylated tRNAs. Discrimination between structurally similar amino acids by these AARSs is believed to operate by a double-sieve principle, wherein a separate editing domain governs hydrolysis on the basis of the size and hydrophilicity of the amino acid side chain. Leucyl-tRNA synthetase (LeuRS) relies on its editing function to correct misaminoacylation of tRNA^(Leu) by isoleucine and methionine. Thr252 of Escherichia coli LeuRS has been shown previously to be important in defining the size of the editing cavity. Here we report the isolation and characterization of three LeuRS mutants with point mutations at this position (T252Y, T252L, and T252F). The proofreading activity of the synthetase is significantly impaired when an amino acid bulkier than threonine is introduced. The rate of misaminoacylation of tRNA^(Leu) by isoleucine and valine increases with the increasing size of the amino acid substituent at position 252, and the noncognate amino acids norvaline and norleucine are inserted efficiently at the leucine sites of recombinant proteins under conditions of constitutive overexpression of the T252Y mutant in E. coli. In addition, the unsaturated amino acids allylglycine, homoallylglycine, homopropargylglycine, and 2-butynylalanine all support protein synthesis in E. coli hosts harboring the mutant synthetase. These results demonstrate that programmed manipulation of the editing cavity can allow in vivo incorporation of novel protein building blocks.

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Tirrell, David A.0000-0003-3175-4596
Additional Information:© 2002 American Chemical Society This work was supported by the NSF Center for the Science and Engineering of Materials at Caltech and by NIH Grant R01-GM62523. Y.T. is supported by a graduate fellowship from the Whitaker Foundation. We thank Dr. Mona Shahgholi and Dr. Gary Hathaway for assistance with typtic digest mass spectrometry. We are grateful to Dr. Anthony Bishop from Prof. Schimmel’s group for his advice on aminoacylation assays. We also thank Prof. Stephen Cusack for providing coordinates of the T. thermophilus LeuRS crystal structure coordinates and P. Wang, I. Carrico, and K. Kirshenbaum for fruitful discussions.
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Whitaker FoundationUNSPECIFIED
Issue or Number:34
Record Number:CaltechAUTHORS:20150108-164212113
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:53349
Deposited By: Donna Wrublewski
Deposited On:09 Jan 2015 23:45
Last Modified:10 Nov 2021 20:00

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