FIG. 3.

Pol I is recruited in the absence of Rrn3p. (A) PIC formation in the absence of Rrn3p. PICs were formed on either WT or Δ−2 templates in extract containing WT Rrn3p (RRN3, lanes 1 and 2), temperature-sensitive Rrn3p (RRN3ts, lanes 3 and 4), or in an extract lacking Rrn3p (ΔRRN3, lanes 5 and 6). After washing, bound proteins were detected by Western blotting. (B) Rescue of transcription activity on PICs lacking Rrn3p. PICs were formed on WT template by incubation with 2 μl of the RRN3ts extract for 45 min. After washing, PICs were incubated either with buffer alone (lanes 2 and 11), a WT extract (2 μl; lane 1), an RPA190ts extract (1, 2, and 4 μl; lanes 3 to 5), affinity-purified yeast Rrn3p (1, 2, and 4 μl; lanes 6 to 8), a ΔRPA190 extract (0.5, 1, and 2 μl; lanes 12 to 14), a ΔRRN7 extract (2 μl; lanes 9 and 15) or a ΔRRN5 extract (5 μl; lanes 10 and 16). Second incubations were also for 45 min followed by washing and resuspension in YTB with NTPs. After 15 min of incubation, transcripts were assayed by primer extension. Partial rescue was obtained with a second extract containing both Pol I and Rrn3p (ΔRRN7; lanes 9 and 15), while an extract containing Pol I, Rrn3p, and CF (ΔRRN5; lanes 10 and 16) rescued fully. (C) Complementation activity of mutant extracts. Extracts were premixed and incubated for 10 min before addition of template. The WT template was added and incubation continued for 45 min followed by NTP addition. Transcription was stopped after 30 min and assayed by primer extension. Volumes of extracts tested were as follows: ΔRPA190, 0.5 μl; RRN3ts, 2 μl; ΔRRN7, 2 μl; ΔRRN5, 5 μl; yRrn3p, 2 μl.