A natural ribozyme with 3',5' RNA ligase activity

Abstract

Using electrophoresis, sequencing and enzymatic digestion, we show that the group I intron from the cyanobacterium Anabaena sp. PCC 7120 catalyzes phosphodiester bond formation using a triphosphate on the 5'-terminal nucleotide, much like protein polymerases and engineered ribozymes. In the process, this ribozyme forms a unique circular RNA that incorporates the exogenous guanosine cofactor added during self-splicing. This finding may have relevance to a prebiotic RNA world and to modern biology.

Figure 1

Reaction pathways catalyzed by group I introns. (a) Self-splicing is followed by the formation of a larger circle (left; this work) or smaller circles (right). The exogenous [α-³²P]GTP used here is shown in red. The subsequently radioactively labeled products are colored in red. (b) Full-length circle formation as described previously,. 5′E, 5′ exon; I, intron; 3′E, 3′ exon.

Figure 2

New circular RNA. (a) Anabaena precursor RNA containing the group I intron after incubation in 0.2 mM GTP, 1.0 μCi ml⁻¹ [α-³²P]GTP, under six reaction conditions (A: 15 mM MgCl₂, 25 mM NaCl, 25 mM HEPES pH 7.5, 32 °C; B: same as A except 42 °C; C: same as A except 1.0 M NaCl; D: same as A except 200 mM MgCl₂; E: 100 mM Mg(OAc)₂, 1.0 M NH₄OAc, 25 mM HEPES pH 7.5, 37 °C; F: 15 mM MgCl₂, 0.5 M KCl, 5 mM DTT, 2 mM spermidine, 40 mM Tris pH 7.5, 50 °C) (from ref. 4). Block arrow, new species. (b) Reaction time-course (condition A), electrophoresis in 0.5× TBE. (c) Same except electrophoresis in 1.0× TBE. (d) Sequencing across the circularization site. N, incorporated residue. (e) Complete nuclease P1 digestion followed by TLC identifies αG incorporated into the circle (lane 1: P4–P6 RNA body-labeled with [α-³²P]GTP and P1-digested; lane 2: gel-purified circle incorporating a single label at the circularization site and P1-digested). (f) Side-by-side time-course experiment as in b using [α-³²P]GTP (left) or [γ-³²P]GTP (right). (g) Complete RNase A plus RNase T1 digestion to determine the regiospecificity at the circularization site, visualized by TLC (lane 1: dinucleotide GpGp control, unlabeled; lane 2: mononucleotide Gp control, unlabeled; lane 3: gel-purified circle incorporating a single label at the circularization site, digested with both RNases). (h) Reaction time-course starting with purified linear intron (condition: A), electrophoresis in 1.0× TBE. (i) Determination of first-order rate constant of circularization. Error bars, s.d. of four experiments (10, 30, 60, 120 min) or two experiments (other data points). Full-length versions of these gels and repeat experiments are shown in Supplementary Figs. 6–9 online.