title: Enzymatic tRNA acylation by acid and alpha-hydroxy acid analogues of amino acids.
abstract: Incorporation of unnatural amino acids with unique chemical functionalities has proven to be a valuable tool for expansion of the functional repertoire and properties of proteins as well as for structure-function analysis. Incorporation of alpha-hydroxy acids (primary amino group is substituted with hydroxyl) leads to the synthesis of proteins with peptide bonds being substituted by ester bonds. Practical application of this modification is limited by the necessity to prepare corresponding acylated tRNA by chemical synthesis. We investigated the possibility of enzymatic incorporation of alpha-hydroxy acid and acid analogues (lacking amino group) of amino acids into tRNA using aminoacyl-tRNA synthetases (aaRSs). We studied direct acylation of tRNAs by alpha-hydroxy acid and acid analogues of amino acids and corresponding chemically synthesized analogues of aminoacyl-adenylates. Using adenylate analogues we were able to enzymatically acylate tRNA with amino acid analogues which were otherwise completely inactive in direct aminoacylation reaction, thus bypassing the natural mechanisms ensuring the selectivity of tRNA aminoacylation. Our results are the first demonstration that the use of synthetic aminoacyl-adenylates as substrates in tRNA aminoacylation reaction may provide a way for incorporation of unnatural amino acids into tRNA, and consequently into proteins.
authors: Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
authors: Alina
authors: A
authors: Owczarek
authors: Mark
authors: M
authors: Safro
authors: Alexey D
authors: AD
authors: Wolfson
mesh_terms: D000143:Acids / Q000737:chemistry / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000378:metabolism*; D049148:Aminoacylation; D001665:Binding Sites; D002855:Chromatography, Thin Layer; D006863:Hydrogen-Ion Concentration; D006880:Hydroxy Acids / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017386:Sequence Homology, Amino Acid; D046249:Transfer RNA Aminoacylation
chemical_list: D000143:Acids; D000596:Amino Acids; D006880:Hydroxy Acids; D000249:Adenosine Monophosphate; D000604:Amino Acyl-tRNA Synthetases