Fig. 3.
Mth Lrp is a transcriptional activator. (A) Transcriptional responses to Mth Lrp (lanes 2–4) and to Mja Ptr2 (lanes 5–7) in the Mja in vitro system (i.e., Mja TBP, TFB, and RNAP) are compared. Single rounds of transcription were carried out at 65°C under the conditions previously optimized for Mja Ptr2 (11), by using the consensus rb2 UAS promoter template in the absence (lane 1) or presence of 200 nM (lanes 2 and 5), 400 nM (lanes 3 and 6), or 800 nM (lanes 4 and 7) regulator. The 84-nt Prb2 run-off transcript and the recovery marker DNA (RM) are indicated on the right. Levels of activation are indicated below each lane. (B) Differential response to Mja Ptr2 by the three methanococcal TBPs, Mja TBP (lanes 1–4), Mth TBP (lanes 5–7), and Mma TBP (lanes 8–12), in conjunction with Mth TFB and RNAP. Single rounds of transcription were carried out at 55°C in the presence of 400 mM NaCl and thermoprotectants (see Materials and Methods), in the absence of Ptr2 (lanes 1, 5 and 9) or in the presence of 250 nM (lanes 2, 6, and 10), 500 nM (lanes 3, 7, and 11), or 1,000 nM (lanes 4, 8, and 12) Ptr2. (C) Differential response to Mth Lrp by the three methanococcal TBPs, analyzed as described in B (the low levels of basal transcription in B and C are inapparent in the printed figure, but were readily detectable in the primary data and quantifiable).