FIGURE 7.

Template-switching of group II intron RTs from 3′-overhang substrates. (A) Template-switching reactions were done with miRNAxs having different 3′-nucleotide residues (lanes A, C, G, U) and initial ³²P-labeled RNA template/DNA primer substrates (IA–P1 RNA/Pc 3′-overhang DNA) having different single nucleotide 3′ overhangs (A, C, G, T, or an equimolar mixture of all four nucleotides [N]; shown schematically below gel). Reactions were with 2 µM TeI4c-MRF RT for 10 min at 60°C in a high-salt reaction medium (450 mM NaCl, 5 mM MgCl₂, 20 mM Tris-HCl [pH 7.5], 1 mM DTT, 1 mM dNTPs), which reduces nontemplated nucleotide addition by the RT. The products were analyzed in a denaturing 20% polyacrylamide gel, which was scanned with a PhosphorImager. Numbers to left of the gel indicate positions of labeled size markers (10-bp ladder). (*) ³²P-label at the 5′ end of primer. (B) Template switching from IA–P1 RNA/Pc DNA with equimolar single-nucleotide 3′ overhangs to an miRNAx with a 3′ phosphorylated C-residue before and after dephosphorylation with T4 polynucleotide kinase (P and DP, respectively); a DNA oligonucleotide of identical sequence (miDNAx); or an miRNAx with a 2′ O-methyl group (CH₃) at its 3′ end.