Figure 3.

Intrinsic endonuclease activity in Tth RNAP EC. (A) Walking of Tth RNAP on an immobilized DNA template. EC14, formed using biotinylated NT DNA strand (NT01) and a 5′ ³²P-labeled RNA primer was immobilized on streptavidin agarose beads, and the RNA primer was extended by several subsequent steps of NTP incorporation and washing at RT (lanes 1–5) and 60°C (lanes 6–10). (B) Nucleolytic activity in Tth RNAP EC16. Tth EC16 was obtained by extension of the RNA primer in EC14 using the walking technique at 60°C (lane 1) and incubated with the CTP (lanes 2 and 3), non-cognate NTPs (lanes 4–6) or without substrates (lane 7) for 5 min at 60°C. (C) EC16 (lane 3) was obtained by extension of the RNA primer in R15/TS35/NT35 by incorporation of [α-³²P]UTP at the 3′ end of the RNA. The complex was then incubated in the presence (lanes 4–7) or absence (lanes 8–11) of 50 μM CTP for 2–40 min at 60°C, and the products of the reaction were analyzed by 23% PAGE electrophoresis. The central portion of the gel was removed for clarity. The position of ³²P-labeled markers—pUpC (lane 1) and pApU (lane 2) are indicated by arrows; * denotes position of the labeled phosphate.