tRNA-dependent amino acid discrimination by Escherichia coli valyl-tRNA synthetase

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Tardif, Keith
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Jack Horowitz
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Biochemistry, Biophysics and Molecular Biology

The Department of Biochemistry, Biophysics, and Molecular Biology was founded to give students an understanding of life principles through the understanding of chemical and physical principles. Among these principles are frontiers of biotechnology such as metabolic networking, the structure of hormones and proteins, genomics, and the like.

The Department of Biochemistry and Biophysics was founded in 1959, and was administered by the College of Sciences and Humanities (later, College of Liberal Arts & Sciences). In 1979 it became co-administered by the Department of Agriculture (later, College of Agriculture and Life Sciences). In 1998 its name changed to the Department of Biochemistry, Biophysics, and Molecular Biology.

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  • Department of Biochemistry and Biophysics (1959–1998)

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Valyl-tRNA synthetase (ValRS) has difficulty discriminating between its cognate amino acid, valine, and structurally similar amino acids, particularly threonine. To minimize translational errors, the enzyme catalyzes a tRNA-dependent editing reaction that prevents accumulation of misacylated tRNA Val. Replacing the universally conserved 3' terminal A of tRNAVal, particularly with pyrimidines (C or U), permits stable misacylation with threonine, alanine, serine, and cysteine. We also observe low levels of aminoacylation of wild type and 3'-end mutants of tRNAVal with isoleucine. ValRS is unable to hydrolytically deacylate misacylated tRNAVal terminating in 3' pyrimidines, but can deacylate tRNAVal terminating in purines (G or A). Evidently, a purine at position 76 of tRNA is necessary for translational editing by ValRS. These misacylated mutant tRNAs act as noncompetitive inhibitors of the aminoacylation reaction. Numerous tRNA mutants have been used to investigate the nucleotides and structural features of tRNA essential for editing. A direct correlation was found between the aminoacylation efficiency of a tRNA and its ability to stimulate the editing reaction, suggesting that editing occurs at the posttransfer step. To identify functional groups on the 3' terminal nucleotide required for function, we replaced the conserved A76 with several different nucleoside analogs and measured their effect on the aminoacylation and editing activity of the tRNA.

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