FIGURE 2.

Intrinsic cleavage activity in E. coli ECs having different lengths of the RNA-DNA hybrid. A, intrinsic cleavage activity of WT E. coli RNAP in ECs assembled on 11-bp (lanes 1–7), 3′ end-mismatched 11-bp (lanes 8–14), 9-bp (lanes 15–21), and 3′ mismatched 9-bp (lanes 22–28) RNA-DNA hybrid scaffolds. E. coli core RNAP was mixed with the scaffold template, and the mixture was incubated at 37 °C for the time indicated. Note that in the scaffolds used the RNA sequence is preserved to exclude its effect on rates of RNA hydrolysis. B, restoration of a normal RNA-DNA hybrid length is due to intrinsic endonucleolytic activity of RNAP. E. coli ECs containing 3′ end-³²P-labeled RNA primer (indicated by an asterisk) were obtained as described under “Experimental Procedures” and incubated at 37 °C for the time indicated, and the products of the reaction were analyzed by 25% PAGE containing 6 m urea (lanes 1–6). RNA dinucleotide primer (ApU) that was 5′ end-labeled with T4 polynucleotide kinase was used as a size marker in this experiment (lane 7).