Figure 3.

Schematic representation of the transcription experiment in vitro. Immobilized RNA polymerase binds to the RNA primer (arrow) annealed to a single-strand DNA oligonucleotide. A complementary non-template DNA is then added to the complex, creating a 3′ overhang for the repeat-containing fragment ligation. The repeat-containing fragment is excised from the plasmid and ligated to the elongation complex. After addition of ATP, CTP and GTP, the elongation complex is stalled upstream from the repeat. Three scenarios are possible after this stalled complex is released in the presence of all four NTPs: (i) successful transcription through the repeat; (ii) transcription arrest within the repeat; and (iii) transcription termination within the repeat.