Transcription on immobilized templates. (A)
Experimental design for the purification of a functional RNAP
II-containing PIC. Promoter-containing templates, immobilized on
magnetic beads, were incubated with nuclear extract, and PICs were
captured by using a magnetic particle concentrator. (B)
Lane 1 shows transcription from WT SL RNA template attached to the
magnetic beads. Lanes 2–4 show transcription from the supernatant
(Sup), wash, and purified PICs on the WT SL RNA template.
(C) Lanes 2, 4, and 6 show the transcripts produced from
purified PICs formed on WT, S-Mut, or Δ-Mut SL templates,
respectively. Lanes 1, 3, and 5 show residual transcriptional activity
in the supernatants. WT SL RNA template (100 ng) was added along with
rNTPs and transcription buffer for supernatant assays.