Self-Splicing of the Group I Intron from Anabaena Pre-tRNA: Requirement for Base-Pairing of the Exons in the Anticodon Stem

In the cyanobacterium Anabaena, the precursor to tRNA^Leu has a 249-nucleotide group I intron inserted between the wobble and second bases of the anticodon; the intron self-splices during transcription in vitro [Xu, M. Q., Kathe, S. D., Goodrich-Blair, H., Nierzwicki-Bauer, S. A., & Shub, D. A. (1990) Science 250, 1566–1570]. By studying splicing of isolated pre-tRNA, we confirm that splicing occurs by the two-step transesterification mechanism characteristic of group I introns, resulting in excision of the intron and accurate ligation of the 5′ and 3′ exons. The first step, guanosine-dependent cleavage of the phosphodiester bond at the 5′ splice site, occurs with k_cat ≃ 14 min⁻¹ and k_cat/K_m = 5 × 10⁴ M⁻¹ min⁻¹ (32 °C, 15 mM MgCl₂), unexpectedly efficient for a small group I intron. (k_cat/K_m is comparable to that of the Tetrahymena pre-rRNA intron, and k_cat is an order of magnitude higher than any previously reported for a group I intron.) The second step, ligation of the exons, is so slow (k = 0.3 min⁻¹) that it is rate-limiting for splicing in vitro except at very low guanosine concentrations. Disruption of the base pairs that make up the anticodon stem of the tRNA dramatically reduces the rate of the first step of splicing, while compensatory mutations that restore base pairing generally restore activity. We suggest that the very short P1 helix of this pre-tRNA, with only three base pairs preceding the 5′ splice site, is unstable without the additional base pairs in the anticodon stem. Thus, splicing of the Anabaena pre-tRNA involves collaboration of the intron and tRNA structures.