Termination facilitates reinitiation.
(A) Time course of transcription. Assays contained
10 ng template DNA (pMrT2/EcoRI)
and partially purified transcription factors. Reactions were started
by addition of nucleotides and samples were taken at the time points
indicated. (B) Sarkosyl sensitivity of TTF-I-mediated transcription
stimulation. Transcription reactions contained 6 µl
S-100 extracts and 30 ng pMrT2/EcoRI
in the absence (lanes 1 and 3) or presence of 5 fmol recombinant
TTFΔN185 (lanes 2 and 4). To prevent
reinitiation, the reactions shown in lanes 3 and 4 contained 0.045% Sarkosyl.
(C) TTF-I is not sufficient for the enhanced synthesis
of terminated transcripts. Transcriptions were performed in a reconstituted
system containing 20 ng pMrT2/EcoRI,
3 ng recombinant UBF, purified TIF-IA, TIF-IB, TIF-IC and two preparations
of Pol I, i.e., H-400 (lanes 1 and 2) or S-300 (lanes 3 and 4).
Where indicated, 5 fmol TTFΔN185 were
added.