Fig. 5. (A) Transcription reactions with either pT7-cont (lanes 1–6) or pT7-nick (lanes 7–12) carried out with the NTPs indicated. Both promoters are synthetic 51 bp duplexes with a –23 to –1 sequence corresponding to the T7 class III consensus. The +1 to +19 non-template strand sequence is GGGAGGACTCCGACAGAGC. pT7-nick differs from pT7-cont in that its template strand is covalently discontinuous and is composed of two oligonucleotides extending from –32 to –5 and –4 to +19. (B and C) Exo III digestion of ITCs formed on either pT7-cont (B) or pT7-nick (C). Reactions allow RNA extension to the lengths indicated above each gel lane. Asterisked RNA lengths indicate that the incoming NTP is present but cannot be incorporated. Positions of the bound and released complexes are indicated as is the length of the undigested template strand at the top of the gel.