TBP domain symmetry in basal and activated archaeal transcription

Abstract

The TATA box binding protein (TBP) is the platform for assembly of archaeal and eukaryotic transcription preinitiation complexes. Ancestral gene duplication and fusion events have produced the saddle-shaped TBP molecule, with its two direct-repeat subdomains and pseudo-two-fold symmetry. Collectively, eukaryotic TBPs have diverged from their present-day archaeal counterparts, which remain highly symmetrical. The similarity of the N- and C-halves of archaeal TBPs is especially pronounced in the Methanococcales and Thermoplasmatales, including complete conservation of their N- and C-terminal stirrups; along with helix H'1, the C-terminal stirrup of TBP forms the main interface with TFB/TFIIB. Here, we show that, in stark contrast to its eukaryotic counterparts, multiple substitutions in the C-terminal stirrup of Methanocaldococcus jannaschii (Mja) TBP do not completely abrogate basal transcription. Using DNA affinity cleavage, we show that, by assembling TFB through its conserved N-terminal stirrup, Mja TBP is in effect ambidextrous with regard to basal transcription. In contrast, substitutions in either its N- or the C-terminal stirrup abrogate activated transcription in response to the Lrp-family transcriptional activator Ptr2.

Fig. 1. TBP stirrup symmetry

(A) A homology model of Mja TBP, shown in ribbon representation with its N- and C-terminal subdomains in red and green, respectively. Positions in the stirrups at which amino acid substitutions were introduced are indicated, and their side chains shown. (B) A list of all Mja TBP variants described in this study and their nomenclature. (C) Sequence alignment of Methanocaldococcus jannaschii (Mja) TBP and its closely related methanogens, Methanococcus vannielii (Mva), Methanococcus maripaludis (Mma), Methanococcus thermolithotrophicus (Mth), Methanococcus aeolicus Nankai (Mae). Also shown are TBPs from Methanopyrus kandleri (Mka), Thermoplasma volcanium GSS1 (Tvo), Thermoplasma acidophilum (Tac), Ferroplasma acidarmanus (Fac), Picrophilus torridus (Pto), Natronomonas pharaonis (Nph), Halobacterium sp. NRC-1 (TBP-E) (Hsp), Haloquadratum walsbyi (Hwa), Halorubrum lacusprofundi (Hla), Haloarcula marismortui (Hma), Sulfolobus solfataricus (Sso), Sulfolobus acidocaldarius (Sac), Hyperthermus butylicus (Hbu), Aeropyrum pernix (Ape), Pyrobaculum aerophilum (Pae), Archaeoglobus fulgidus (Afu), Methanothermobacter thermoautotrophicus (Mau), Thermococcus kodakaraensis (Tko), Pyrococcus furiosus (Pfu), Candidatus Korarchaeum cryptofilum (CKc), Saccharomyces cerevisiae (Yeast) and Homo sapiens (Human). Identical, similar and non conserved residues are shaded in yellow, cyan and red, respectively.

Fig. 2. Basal transcriptional activity of Mja TBP stirrup mutants

(A) Single rounds of basal transcription at the strong Pfu gdh promoter were carried out in the Mja in vitro system at 65°C (see Experimental procedures), in the absence of TBP (lane 1) or in the presence of 10, 20, 40, or 80 nM wild type TBP (lanes 2–5, respectively), TBP nAAA (E35A-E37A-Q38A) (lanes 6–9), TBP cAAA (E126A-E128A-Q129A) (lanes 10–13), or TBP ncAAA (combining all six alanine substitutions; lanes 14–17). The 83-nt P_gdh run-off transcript and the recovery marker DNA (RM) are indicated on the right. (B) Levels of basal transcription; data from (A), quantified (A.U.: arbitrary units).

Fig. 3. Affinity cleavage of the gdh T/A-box by Mja TBP-OP derivatives

(A) The sequence of the 75-bp gdh promoter probe is shown, with the T/A-box and the start site of transcription boxed. The alkylated TBP derivatives WT-OP, nAC^OPA and cAC^OPA were bound individually, at 65°C, to the DNA probe 5′-end labeled on the top (non-transcribed) strand (B) or the bottom (transcribed) strand (C), in the absence or in the presence of TFB, as indicated above each lane. CuSO₄, H₂O₂, and mercaptopropionic acid were then added to initiate DNA cleavage, as described in Experimental procedures. WT-OP refers to wild type TBP that was alkylated as a negative control. Also shown are the untreated DNA probe (P) and the A+G chemical sequencing ladder. In the diagram on the left, the “Downstream” arrow points from the T/A box toward the transcriptional start. (D) Schematic illustration of promoter complexes containing the nAC^OPA and cAC^OPA TBP derivatives. The specified TBP orientations correspond to those that would result in the cleavage patterns observed in (B) and (C). N and C refer to the pseudosymmetrical halves of TBP.

Fig. 4. Functional dominance of the gdh promoter BRE

(A) Cartoon of the mutant gdh promoter engineered with an additional initiator element (open arrowhead) placed upstream of the BRE (gray-shaded box). The T/A box and the gdh initiator element are shown as an open box and a filled arrowhead, respectively. (B) Single rounds of basal transcription at the wild type (lanes 1-2) or mutant Pfu gdh promoter (lanes 3-4) were carried out in the presence of 40 nM TFB and 80 nM TBP, either wild type (lanes 1 and 3) or cAAA (E126A-E128A-Q129A) (lanes 2 and 4). The 83-nt P_gdh run-off transcript and the recovery marker DNA (RM) are indicated on the right. Also indicated is the expected position of the presumptive 68-nt run-off product of transcription in the upstream (anti-gdh) orientation. The DNA size marker indicated on the left was provided by the A+G chemical sequencing ladder shown in Fig. 3B.

Fig. 5. Transcriptional response to Ptr2 by TBP stirrup mutants

(A) P_rb2 DNA template with an optimized UAS (Ouhammouch et al., 2005) was incubated in the absence of (lanes 1, 4, 7 and 10), or presence of 400 nM (lanes 2, 5, 8 and 11), or 800 nM (lanes 3, 6, 9 and 12) Ptr2 and used in a single-round transcription assay driven by wild type or mutant Mja TBP (20 nM) indicated above each lane. The 84-nt P_rb2 run-off transcript and the recovery marker DNA (RM) are indicated on the right. (B) Varying concentrations of wild type TBP and mutant nAAA (E35A-E37A-Q38A) were compared for ability to respond to activation by Ptr2. Single-round transcription was carried out in the absence (lanes 1-3 and 7-9) or presence of 400 nM (lanes 4-6 and 10-12) Ptr2, and driven by varying concentrations of the indicated TBP: 40 nM (lanes 1, 4, 7 and 10), 80 nM (lanes 2, 5, 8 and 11), or 160 nM (lanes 3, 6, 9 and 12). The fold-activation elicited by Ptr2 at each TBP concentration is indicated below the panel.