Figure 6. Template switching of LtrA from the 5′ end of the Ll.LtrB intron RNA to exon 1 DNA or RNA.

The Ll.LtrB intron RT (LtrA protein; 40 nM) was incubated with artificial substrates corresponding to the 5′ end of Ll.LtrB intron (Ll.LtrB RNA; 40 nM) with an annealed 5′-³²P-labeled DNA primer c (Pri c; 44 nM) in presence of exon 1 (E1) DNA or RNA (40 nM; black and red, respectively), as diagrammed in schematics to the left of the gel. The substrates were incubated with dNTPs (200 µM) in reaction medium containing 450 mM NaCl, 5 mM MgCl₂, 20 mM Tris-HCl, pH 7.5, and 1 mM DTT for 30 min at 30°C. After terminating the reaction by extraction with phenol-CIA, the products were analyzed in a denaturing 15% polyacrylamide gel. Lanes (1) and (2) ³²P-labeled Pri c incubated without and with LtrA, respectively; (3) and (4) LtrA incubated with ³²P-labeled Pri c and E1 DNA or RNA, respectively; (5) and (6) LtrA incubated with Ll.LtrB RNA with annealed ³²P-labeled Pri c and E1 DNA or RNA, respectively; (7–9) LtrA incubated with Ll.LtrB RNA with annealed ³²P-labeled Pri c and E1 DNA or RNA with annealed complementary DNA oligonucleotides to leave a blunt end (exon 1 AS) or a 5′-bottom-strand overhang (exon 1 AS+9). Bands excised for sequencing (Figure 7) are indicated in the gel. In the schematics, DNA and RNA oligonucleotides are shown in black and red, respectively; LtrA is shown as a gray oval; and the direction of cDNA synthesis is indicated by a green arrow. The numbers to the right of the gel indicate the positions of 5′-end labeled size markers (10-bp DNA ladder, Invitrogen).