tRNA 3'-Amino-Tailing for Stable Amino Acid Attachment

Abstract

Amino acids are attached to the tRNA 3'-end as a prerequisite for entering the ribosome for protein synthesis. Amino acid attachment also gives tRNA access to non-ribosomal cellular activities. However, the normal attachment is via an ester linkage between the carboxylic group of the amino acid and the 3'-hydroxyl of the terminal A76 ribose in tRNA. The instability of this ester linkage has severely hampered studies of aminoacyl-tRNAs. Although the use of 3'-amino-3'-deoxy A76 in a 3'-amino-tailed tRNA provides stable aminoacyl attachment via an amide linkage, there are multiple tailing protocols and the efficiency of each relative to the others is unknown. Here we compare 5 different tailing protocols in parallel, all dependent on the CCA-adding enzyme ((CTP(ATP): tRNA nucleotidyl transferase; abbreviated as the CCA enzyme) to exchange the natural ribose with the modified one. We show that the most efficient protocol is achieved by the CCA-catalyzed pyrophosphorolysis removal of the natural A76 while at the same time adding the appropriate ATP analog to synthesize the modified 3'-end. This protocol for 3'-amino-tailing affords quantitative and stable attachment of a broad range of amino acids to tRNA, indicating its general utility for studies of aminoacyl-tRNAs in both canonical and non-canonical activities.