In Vitro Engineering Using Acyl-Derivatized tRNAs
Incorporation of fluorescently labeled or otherwise modified amino acids into proteins is potentially useful in a variety of ways, including monitoring the folding of the nascent protein chain, measuring posttranslational conformational changes and binding of substrates, and determining structure-function relationships. One of the most successful techniques for incorporating fluorescent amino acids into proteins has been to use aminoacyl-tRNAs in which the α-amino group has been acylated with a fluorescent moiety producing an N-acylaminoacyl-tRNA derivative (1). These derivatives have been used in then own right to study tRNA binding sites on the ribosome (2). Such derivatives, with their acylated amino groups, can only serve as donors in protein synthesis and thus can only be incorporated at the N-terminus. Nevertheless they have been very useful in studying the poly (U)1-directed synthesis of polyphenylalanine, polyserine, and polyalanine, the latter two using synthetic tRNAs whose anticodons had been changed to AAA (3). By incorporating coumarin-labeled phenylalanine or alanine at the N-terminus of such nascent chains, we were able to study by fluorescence, the interactions of the nascent chains with the ribosome as a function of chain length (4,5). More recently, using an efficient coupled transcription/translation system from Escherichia coli (6), we have incorporated coumarin-labeled methionine from tRNAf Met into several proteins. The fluorescent probe has been instrumental in studying interactions of those proteins with molecular chaperones (7).
KeywordsHigh Pressure Liquid Chromatography Chloramphenicol Acetyl Transferase Succinimidyl Ester Circular Plasmid Mercaptoacetic Acid