Two reactions of Haloferax volcanii RNA splicing enzymes: Joining of exons and circularization of introns

Splicing of truncated elongator pre-tRNA^Met. Sequences of the precursor (A) and its circularized intron (B). The anticodon is highlighted in A. Arrows indicate the splice sites within the BHB motif in A and the junction in B. Phosphates derived from [α-³²P]ATP labeling of the precursor are denoted by asterisks in A and B. Products of endonuclease (C, lane 1) followed by ligase (C, lane 2) using [α-³²P]ATP-labeled precursor are separated on a 50-cm long, denaturing 15% gel. (P) Precursor; (LI) linear intron; (5′+3′) two exons together; (3′) only 3′ exon; (LE) ligated exons; (CI) circular intron; (XC) xylene cyanol. The gel-eluted precursor and its intron products (shown in C) were digested with nuclease P1 and resolved by TLC (D–F) using solvent a in the first dimension and solvent b in the second dimension. (D) Precursor (P). (E) Linear intron (LI). (F) Circular intron (CI). Outlines indicate the positions of nonradioactive markers in D–F.