Loss of positional specificity in the aminoacylation of Escherichia coli tRNAGly

• Published in: The Journal of biological chemistry Vol. 258; no. 19; pp. 11745 - 11750
• Main Authors: Ehrenfeld, G M, Francis, T A, Hecht, S M
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 10-10-1983; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acyl-tRNA Synthetases - metabolism; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Enzymes and enzyme inhibitors; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Glycine-tRNA Ligase - metabolism; Hydrogen-Ion Concentration; Kinetics; Ligases; Magnesium - pharmacology; Nucleic Acid Conformation; RNA, Transfer, Amino Acyl - genetics; Glycyl-tRNA synthetase; Molecular interaction; Aminoacylation; Specificity; Escherichia coli; Conformation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)44292-X

The positional specificity in the aminoacylation of Escherichia coli tRNAGly by its cognate aminoacyl-tRNA synthetase has been studied using tRNAGlys terminating in 2'- or 3'-deoxyadenosine under conditions believed to alter tRNA conformation. Although E. coli tRNAGly terminating in 3'-deoxyadenosine has been reported not to be a good substrate for activation by the homologous glycyl-tRNA synthetase, by systematic variation of the conditions employed for aminoacylation it was possible to activate this tRNA to essentially the same extent as unmodified tRNAGly. Activation of tRNAGly terminating in 3'-deoxyadenosine was carried out optimally at 45 degrees C in an incubation mixture containing 0.3-0.4 M NaCl; 10% methanol, ethanol, and dimethyl sulfoxide were found to facilitate activation of the modified tRNA. Interestingly, the conditions employed to enhance activation of this modified tRNAGly had no effect on the activation of unmodified tRNAGly or tRNAGly terminating in 2'-deoxyadenosine. These experiments afford insight into the activation of tRNAGly by glycyl-tRNA synthetase and provide facile access to positionally defined, isomeric glycl-tRNAGlys.