FIG. 2.

Cleavage (and extension) of the Tf1-derived RNA sequence by Tf1 RT and HIV-1 RT. The in vitro-synthesized 365-nt RNA was 5′-end labeled with [γ-³²P]ATP (see Materials and Methods). This RNA was incubated with or without 100 ng of Tf1 RT or HIV-1 RT, and each reaction was conducted in the absence of or with 5 mM MgCl₂ or 0.5 mM MnCl₂ (in the presence or the absence of 50 μM dNTPs). After incubating for 10 min at 37°C, the reaction products were resolved by high-voltage and high-resolution electrophoresis through 12% polyacrylamide gels with 6 M urea. All reaction products contained the RNA substrate and other components, as shown in the following gel lanes: 1, the 365-nt RNA substrate alone; 2, with Mg²⁺; 3, with Mn²⁺; 4, with Tf1 RT; 5, with Tf1 RT and Mg²⁺; 6, with Tf1 RT and Mn²⁺; 7, with Tf1 RT, Mn²⁺, and all four dNTPs; 8, with Tf1 RT, Mn²⁺, dATP, dCTP, and dGTP; 9, with Tf1 RT, Mn²⁺, dATP, dCTP, and dTTP; 10, with HIV-1 RT; 11, with HIV-1 RT and Mg²⁺; and 12, with HIV-1 RT and Mn²⁺. To precisely localize the position of the produced self-primer, a 23-nt 5′-end-labeled synthetic marker RNA was used (indicated by an arrow). The position of the 18-nt product, generated by HIV-1 RT (in the presence of Mn²⁺), was determined from the molecular ladder of the partially cleaved 23-nt RNA marker.