FIG. 3.
Efficiency of tRNA3Lys-primed minus-strand ssDNA synthesis in cell-free assays. The efficiencies of synthesis of full-length DNA with WT and M230L mutant RTs were compared in time course experiments. (A) Graphic representation of the cell-free system used for the synthesis of full-length minus-strand ssDNA. The PBS RNA template used in this system consists of 258 nt at the 5′ end of the HIV-1 genome, which contains the R, U5, and PBS regions. (B) Synthesis of full-length (FL) DNA by recombinant WT and mutant enzymes monitored in time course experiments. The reactions were initiated by the addition of 10 μM dNTPs and were monitored by measurement of the incorporation of [α-32P]dCMP into the extending DNA strand. The reactions were stopped at different time points during a period of 45 min. Time point zero indicates samples taken at 20 s after initiation. The full-length DNA product and the +1, +3, and +5 pausing sites are shown on the right.