Architecture and transcription of RNA–DNA
dumbbell by RNA pol II with and without TFIIS. (A)
Schematics of the nucleic acid framework in the elongation bubble and
its minimalistic mimic dumbbell. Arrows indicate transcription
direction, and solid and open circles represent deoxy- and
ribonucleotides, respectively. In the sequence of the dumbbell
presented, the ribonucleotides are shaded. Several dumbbells in which
the nature of the 3′-terminal ribonucleotide X and deoxynucleotide Y
were varied are shown. (B) Elongation and cleavage of DB1 by
pol II with and without TFIIS. RNA synthesis was carried out by pol II
without preincubation (0 min) or after preincubation (30 min) with DB1
followed by addition of indicated NTP. The template-dependent
elongation of the 41-nt dumbbell DB1, in the presence of four NTPs and
pol II generates a run-off 58-nt RNA–DNA duplex. The ribonuclease
activity of pol II cleaves DB1 into 39-nt dumbbell. In the RNA cleavage
reaction, NTPs were omitted.