An expanding family of archaeal transcriptional activators

Abstract

Transcriptional regulation in the archaea involves a mosaic of DNA-binding proteins frequently (although not exclusively) of bacterial type, modulating a eukaryal-type core transcription apparatus. Methanocaldococcus jannaschii (Mja) Ptr2, a homologue of the Lrp/AsnC family of bacterial transcription regulators that are among the most widely disseminated archaeal DNA-binding proteins, has been shown to activate transcription by its conjugate hyperthermophilic RNA polymerase. Here, two in vitro systems have been exploited to show that Ptr2 and a Lrp homologue from the thermophile Methanothermococcus thermolithotrophicus (Mth) activate transcription over a approximately 40 degrees C range, in conjunction with their cognate TATA-binding proteins (TBPs) and with heterologous TBPs. A closely related homologue from the mesophile Methanococcus maripaludis (Mma) is nearly inert as a transcriptional activator, but a cluster of mutations that converts a surface patch of Mma Lrp to identity with Ptr2 confers transcriptional activity. Mja, Mth, and Mma TBPs are interchangeable for basal transcription, but their ability to support Lrp-mediated transcriptional activation varies widely, with Mja TBP the most active and Mth TBP the least active partner. The implications of this finding for understanding the roles of TBP paralogues in supporting the gene-regulatory repertoires of archaeal genomes are briefly noted.

Fig. 1.

Two methanococcal homologues of Ptr2. (A) Sequence alignment of Mja Ptr2, Mth Lrp, and Mma Lrp. Conserved, similar, and differing residues are shaded in yellow, blue, and red, respectively. The putative DNA-binding, linker, and effector domains are indicated above the alignment. (B) Homology model of Mma Lrp, shown in surface representation as a dimer of green and yellow protomers. Positions of sequence divergence from Mja Ptr2 are colored as in A, with putative activation determinants labeled. Also shown are the amino acid substitutions that generate Mma Lrp mutants 1, 2, and 3.

Fig. 2.

Specific binding of Mth and Mma Lrp to the consensus UAS of the rb2 promoter. The DNA probe, 5′-end-labeled on the nontranscribed (nontemplate) strand, was incubated at 55°C (A) or 37°C (B) for 20 min in the absence of Lrp (lane 3 of each panel) or with increasing concentrations of Mth Lrp (A) or Mma Lrp (B) (indicated above each panel and specified throughout for the monomer) and subjected to ·OH cleavage for 30 s at the same temperature. ·OH cleavage products within the T/A box and the two Ptr2 consensus binding sites are boxed in lane 3 and identified at the left of each panel. Also shown are the untreated DNA probe (P) and G and A + G chemical-sequencing ladders.

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 P_rb2 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).

Fig. 4.

The three methanococcal TBPs are equally active in basal transcription. (A) Single rounds of basal transcription at a strong promoter (an up-mutant ptr1 promoter with an improved BRE) were carried out as described for Fig. 3 B and C, in the absence of TBP (lane 1) or in the presence of 5, 10, 20, 40, or 80 nM Mja TBP (lanes 2–6, respectively), Mth TBP (lanes 7–11), or Mma TBP (lanes 12–16). (B) Levels of basal transcription; data from A, quantified.

Fig. 5.

Converting Mma Lrp into a potent transcriptional activator. (A) Comparison of activation levels elicited by 250, 500, or 1,000 nM Mja Ptr2 (lanes 2–4, respectively), wild-type Mma Lrp (lanes 5–7), mutant 1 (lanes 8–10), mutant 2 (lanes 11–13), and mutant 3 (lanes 14–16), in conjunction with Mja TBP, Mth TFB, and Mth RNAP. Single rounds of transcription were carried out at 37°C in the presence of 250 mM NaCl (and in the absence of thermoprotectants). (B) Levels of activation; data from A.(C) Transcriptional response to wild-type Mma Lrp by the three methanococcal TBPs. Single rounds of transcription were carried out as in A. (D) Transcriptional response to Mma Lrp mutant 1 by the three methanococcal TBPs.