Fig 2.
trans-priming from the TP and RT domains by purified MiniRT2. Purified MiniRT2, TP, and RT domains were mixed together as indicated for protein priming in the presence of [α-32P]dGTP and the DHBV ε RNA. The RT proteins used were SUMO-MiniRT2 refolded (lane 4) or natively purified (lanes 3 and 8 to 10), natively purified GST-MiniRT2 (lanes 5 to 7), His-MiniRT2 (lanes 11 to 14), and His-MiniRT2-Y96F (lanes 15 to 18). The individual domains used were His-tagged and natively purified (lanes 1, 3, 13, and 17) or refolded (lanes 2, 4, 6, and 9) TP or TP-Y96F (lanes 14 and 18), natively purified GST-TP (lanes 7 and 10), and His-tagged RT domain natively purified (lanes 1, 12, and 16) or refolded (lane 2). All priming reactions were conducted in TMnNK. Priming reaction products were resolved by SDS-PAGE and detected by autoradiography. The labeled RT proteins and domains, as a result of cis- or trans-priming, are indicated. All reactions used 1 pmol of proteins/domains, except those shown in lanes 11 to 18, which used 4 pmol of His-TP or His-RT.