Figure 2.

The AnCOB intron encodes a maturase that directly assists RNA splicing. (A) Group I introns react by two sequential transesterification reactions: First, a guanosine attacks the 5′ splice site and is transferred to the first base of the intron. The exposed 3′OH end of the upstream exon (5′ exon) then attacks the 3′ splice site, releasing the intron and leaving the exons ligated (58). PRE, precursor; I3E, intron + 3′ exon splicing intermediate; I, excised intron; LE, ligated exons. The 5′ exon is short and is not shown. Its presence exactly mirrors that of the I3E molecule, being generated during 5′splice-site cleavage and disappearing as RNA molecules complete the second step. (Lane a) Prior to mixing with RNA, the protein extract was treated with 2 μg/μl of proteinase K for 30 min at 37°C. (lanes b and c) The pre-RNA was incubated as above (5 mM MgCl₂) with extracts purified by Ni⁺⁺ affinity chromatography (as for the AnCOB protein) from BL21DE3 either containing just the pET28b vector (lane b) or expressing the endonuclease encoded by a different intron, AnOX2 (37) (lane c). (B) The rate of the first splicing step was determined as described in Materials and Methods. ▴, 25 mM Mg²⁺, no maturase; ⧫, 5 mM Mg²⁺ + maturase; ▪, 25 mM Mg²⁺ + maturase.