FIG. 3.

RNase H cleavage sites in the vicinity of the −1/+1 cleavage site on the 79-nt MLV RNA containing an internal PPT. Either 5′ end-labeled (lanes 1 to 4) or 3′ end-labeled (lanes 5 to 9) 79-nt MLV RNA (containing M-MuLV sequence from −54 to +17) was annealed to template 2 in substrate IV (schematic at top), and these hybrids were incubated with a 10:1 (lanes 2 and 6) or 50:1 (lanes 3,7, and 8) molar ratio of wild-type reverse transcriptase/substrate for 15 or 30 s as indicated. Substrate incubated without enzyme is shown in lanes 1 and 5, and substrate digested with nuclease P1 to generate marker bands is shown in lanes 4 and 9. Samples were analyzed in a 20% sequencing gel and visualized using a PhosphorImager. Shown vertically at the left and right is the region of sequence containing the PPT (boxed) from the plus-sense M-MuLV genome (positions 7798 to 7832). In the sequence, the positions of observed cleavage sites are indicated with arrows. For prominent sites, the base positions bordering each site are given opposite the arrows and the arrows are connected to corresponding bands by dashed lines. For both 5′ and 3′ end-labeled substrates, positions of bands resulting from cleavage at the −23/−22 site are indicated in the center. In lanes 6 to 8, bands resulting from 5′ end-directed cleavages are indicated with a bar and line at right (5′ end direct.).