Architecture and folding mechanism of the Azoarcus group I pre-tRNA

Self-splicing RNAs must evolve to function in their specific exon context. The conformation of a group I pre-tRNA^ile from the bacterium Azoarcus was probed by ribonuclease T₁ and hydroxyl radical cleavage, and by native gel electrophoresis. Biochemical data and three-dimensional models of the pre-tRNA showed that the tRNA is folded, and that the tRNA and intron sequences form separate tertiary domains. Models of the active site before steps 1 and 2 of the splicing reaction predict that exchange of the external G-cofactor and the 3′-terminal G is accomplished by a slight conformational change in P9.0 of the Azoarcus group I intron. Kinetic assays showed that the pre-tRNA folds in minutes, much more slowly than the intron alone. The dependence of the folding kinetics on Mg²⁺ and the concentration of urea, and RNase T₁ experiments showed that formation of native pre-tRNA is delayed by misfolding of P3-P9, including mispairing beween residues in P9 and the tRNA. Thus, although the intron and tRNA sequences form separate domains in the native pre-tRNA, their folding is coupled via metastable non-native base-pairs. This could help prevent premature processing of the 5′ and 3′ ends of unspliced pre-tRNA.