Jonathan J. Burbaum

[25] Transfer RNA with double identity for in Vitro kinetic modeling of transfer RNA identity in Vivo

Publisher Summary Because there are approximately 60 different transfer RNAs (tRNAs) and 20 aminoacyl-tRNA synthetases in the cytoplasm of a prokaryotic or eukaryotic cell, the aminoacylation of a particular tRNA by its cognate enzyme is only one of approximately 1200 potential amino acid-tRNA combinations. These combinations are accessible in principle, because tRNAs have a similar L-shaped three-dimensional structure and have conserved nucleotides that stabilize the spatial arrangement of bases and phosphate groups. In addition to specific editing reactions that prevent or correct misacylations, the high accuracy of the code in vivo is responsible in significant part to binding interactions that sequester tRNAs with their cognate aminoacyl-tRNA synthetases, and thereby diminish the pool of each that is available to interact with a noncognate partner. This possibility was demonstrated in vitro with Escherichia coli isoleucine–tRNA synthetase (ligase), which aminoacylates tRNA and misacylates tRNA. Methods for measuring the intracellular concentrations of synthetases, tRNAs, and amino acids in E. coli are described in the chapter.