New biochemical insights of CCA enzyme role in tRNA maturation and an efficient method to synthesize the 3′-amino-tailed tRNA

Abstract

The maturation of tRNA and its quality control is crucial for aminoacylation and protein synthesis. The CCA enzyme, also known as tRNA nucleotidyltransferase, catalyzes the addition or repair of CCA at the 3′-terminus of tRNAs to facilitate aminoacylation. Structural studies of CCA enzyme in complex with ATP and CTP suggested that adding CCA at the 3′-terminus of tRNAs is a sequential process [1–4]. However, there are many inconsistent results of CCA addition from the biochemical studies, which raise the ambiguity about the CCA enzyme specificity in vitro [5–7]. On the other hand, there are no effective methods for preparing the 3′-amino-tailed tRNA to provide a stable amide linkage, which is vital to make homogeneous samples for structural studies of stalling peptides to understand ribosome mediated gene regulation [7–11]. In this study, we examined the functional specificity of the Class II CCA enzyme from E. coli, and optimized the benchmark experimental conditions to prepare the 3′-NH2-tRNA using the CCA enzyme. Our results suggest that the CCA enzyme has a specific ability to catalyze the CCA addition/repair activity within the stoichiometric range of the reactants, and excess amounts of nucleotides lead to non-specific polymerization of the tRNA. Further, we developed an efficient method for synthesizing 3′-amino tRNA, which can facilitate stable aminoacyl/peptidyl-tRNA preparation.