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. Author manuscript; available in PMC: 2010 Jan 1.
Published in final edited form as: Mol Microbiol. 2008 Nov 4;71(1):123–131. doi: 10.1111/j.1365-2958.2008.06512.x

TBP Domain Symmetry in Basal and Activated Archaeal Transcription

Mohamed Ouhammouch 1,*, Winfried Hausner 2, E Peter Geiduschek 1
PMCID: PMC2670189  NIHMSID: NIHMS97737  PMID: 19007415

Summary

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.

Keywords: TATA-binding protein, Transcription factor B, RNA polymerase, Ptr2, Archaea

Introduction

The TATA-box binding protein (TBP) is the central component directing the assembly of transcription preinitiation complexes (PICs) in archaea and eukaryotes (Burley, 1996, Hausner et al., 1996). Archaeal promoters consist of a T/A-rich TATA-like element recognized by the archaeal TBP, and located immediately downstream of the BRE [the binding site for the eukaryotic TFIIB-like initiation factor TFB]. Just as eukaryotic TBP and TFIIB recruit RNA polymerase (pol) II, archaeal TBP and TFB position archaeal RNA polymerase (RNAP) over a simple initiator element that marks the transcriptional start site (reviewed in (Thomm, 1996, Bell & Jackson, 2001, Geiduschek & Ouhammouch, 2005)).

Crystal structures of both eukaryal TBP-TFIIB (Nikolov et al., 1995, Tsai & Sigler, 2000) and archaeal TBP-TFB complexes (Littlefield et al., 1999) bound to short TATA box-containing promoter fragments reveal a highly distorted promoter in which the TATA box is partially unwound and bent toward the major groove. In these structures, TBP interacts with the minor groove of the DNA primarily through nonspecific hydrophobic contacts with the bases, giving rise to a TBP-TATA box interface with nearly perfect symmetry. In archaea, the unidirectionality of PIC assembly and transcription is enforced by the BRE, a consensus sequence element of at least six or seven base pairs immediately upstream of the T/A-box and contacted by TFB (Qureshi & Jackson, 1998, Bell et al., 1999). Because the very same interaction has been described for eukaryotic RNA pol II promoters, TFIIB/BRE interaction is also thought to dictate the polarity of RNA pol II transcription (Lagrange et al., 1998, Tsai & Sigler, 2000).

In the crystal structures of both archaeal and eukaryal ternary complexes, TBP is bound in the same orientation relative to the start site of transcription, that is with its C-terminal stirrup on the upstream side of the TATA box. Along with the similarly upstream-facing helix H′1, the C-terminal stirrup of TBP contributes most of the specific protein-protein contacts at its interface with TFIIB (Nikolov et al., 1995, Tsai & Sigler, 2000), and TFB (Littlefield et al., 1999). Specific substitutions in the C-terminal stirrup of both mammalian and yeast TBP abrogate their ability to recruit TFIIB to the promoter complex and direct basal transcription (Bryant et al., 1996, Tang et al., 1996, Lee & Struhl, 1997).

In this paper, 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 a DNA affinity cleavage method, we show that these TBP variants are able to assemble TFB and direct basal transcription through their conserved N-terminal stirrup. We also show that substitutions in either the N-terminal or the C-terminal stirrup of Mja TBP abrogate activated transcription in response to the Lrp-family transcriptional activator Ptr2.

Results

TBP stirrup symmetry and basal transcription

The saddle shape of TBP is formed by its two direct-repeat subdomains, each with five β-strands and two α-helices. These two subdomains are connected in such a way that the whole molecule presents a pseudo-two-fold symmetry (Fig. 1A). TBP is thought to have evolved from a (symmetrical) homodimer by gene duplication. Collectively, eukaryotic TBPs have diverged farther from their present-day archaeal counterparts, which remain highly symmetrical. Overall, TBPs from Mja and its closely related Methanococcales Methanococcus maripaludis (Mma), Methanothermococcus thermolithotrophicus (Mth) and Methanococcus vannielii (Mva) stand out as the most symmetrical of all known TBPs. The N- and C-terminal halves of Mja TBP share ∼ 69% overall similarity, compared to 51% for Sulfolobus acidocaldarius, 48% for Pyrococcus furiosus (Pfu), 42% for yeast and 39% for human TBP. Nowhere is this sequence conservation more striking than at their N- and C-terminal stirrups, which are completely conserved (Fig. 1C), a feature also shared by the TBPs from Methanopyrus kandleri and several Thermoplasmatales (Thermoplasma volcanium, Thermoplasma acidophilum, Ferroplasma acidarmanus and Picrophilus torridus). As already mentioned, the upstream-facing helix H′1 and the C-terminal stirrup of TBP form the main interface with TFB/TFIIB (Nikolov et al., 1995, Tsai & Sigler, 2000). The near symmetry of Mja TBP and of its T/A box binding site raises questions about its orientation in the promoter complex.

Fig. 1. TBP stirrup symmetry.

Fig. 1

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(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.

To address this question and gain insight into the functional significance of strict sequence conservation in the stirrups, we generated a set of Mja TBP variants with alanine substitutions at critical residues: E126A-E128A-Q129A in the C-terminal stirrup (cAAA), and at the corresponding residues in the N-terminal stirrup (nAAA: E35A-E37A-Q38A) (Figs 1A and 1B). It is worth noting that point mutations at individual residues in the C-terminal stirrup of both mammalian and yeast TBP corresponding to the substitutions in Mja TBP mutant cAAA severely affect their ability to assemble TFIIB and direct basal transcription by RNA pol II ((Bryant et al., 1996, Tang et al., 1996, Lee & Struhl, 1997); see Discussion). The ability of mutants nAAA and cAAA to support basal transcription was tested in the Mja in vitro system (Ouhammouch et al., 2003) at the well-characterized strong Pyrococcus furiosus (Pfu) gdh promoter (Hethke et al., 1996). As shown in Fig. 2A, mutant nAAA and the wild type TBP are comparably active on this strong promoter (compare lanes 6-9 to lanes 2-5). TBP cAAA, on the other hand, retained nearly 50% of the activity of wild type protein (Fig. 2A, lanes 10-13, and Fig. 2B), while TBP ncAAA (E35A-E37A-Q38A-E126A-E128A-Q129A), combining the alanine substitutions in nAAA and cAAA, was found to be transcriptionally inert (Fig. 2A, lanes 14-17). Hydroxyl radical footprinting experiments showed that TBP cAAA is strongly defective in its ability to bind DNA on its own, and that this defect is only partially relieved by the presence of TFB (results not shown), which could explain, at least in part, its observed transcriptional defect.

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

Fig. 2

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(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 Pgdh 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).

Taken together, these data suggest a possible functional redundancy between the N- and C-terminal stirrups of Mja TBP in specifying an assembly epitope for TFB.

DNA affinity cleavage and orientation of TBP in ternary complexes

The data presented above forced a re-examination of the geometry of assembly of PICs in Mja, and the widely-held view of the C-terminal repeat of TBP as the unique assembly platform for TFIIB-related factors across the archaeal and eukaryal kingdoms. For this, we turned to a previously described DNA affinity cleavage technique that provides a direct read-out of the orientation of TBP on the TATA box (Cox et al., 1997, Kays & Schepartz, 2000). Two new Mja TBP variants were generated: nACA (E35A-E37C-Q38A) containing a reactive cysteine at position 37 within the N-terminal stirrup, and cACA (E126A-E128C-Q129A), containing a reactive cysteine at position 128 within the C-terminal stirrup (Figs 1A and 1B). The basal transcription activities of cysteine mutants nACA and cACA at the gdh promoter were indistinguishable from those of nAAA and cAAA, respectively (Fig. S1, Supporting information). These proteins were modified with 5-iodoacetamido-1,10-phenanthroline (IAAOP), a 1,10-phenanthroline (OP) derivative that specifically alkylates cysteine residues (Chen & Sigman, 1987). In the presence of Cu+, a reducing agent, and hydrogen peroxide, these TBP-OP proteins form a reactive Cu complex capable of cleaving DNA through abstraction of hydrogen from the deoxyribose backbone. Because Mja TBP contains 2 additional cysteine residues, wild type TBP was derivatized with IAAOP in parallel with mutants nACA and cACA, and used as a control for all DNA affinity cleavage experiments.

According to the orientation of TBP in the Pyrococcus woesei TBP-TFB-DNA ternary complex crystal structure (Littlefield et al., 1999), the thiol group of TBP nACA cysteine 37 would be located in the DNA minor groove. The ternary complex formed by TFB and TBP nACOPA bound to the gdh promoter in that orientation should generate DNA cleavage confined to the downstream edge of the T/A-box. As expected, nACOPA cleaved DNA predominantly downstream of the T/A-box in ternary complexes, 5′ end-labeled either on the top strand (Fig. 3B, lane 5) or on the bottom strand (Fig. 3C, lane 5). The striking outcome of this experiment was finding that TBP cACOPA generates a similar cleavage pattern downstream of the T/A-box, on the top strand (Fig. 3B, lane 7) as well as the bottom strand (Fig. 3C, lane 7), indicating a ternary complex geometry in which TBP is bound to the DNA in the opposite orientation, that is, with its C-terminal subdomain facing the downstream edge of the T/A-box (Fig. 3D). In the absence of TFB, TBP cACOPA generated a detectable level of cleavage upstream of the T/A-box, on the top (Fig. 3B, lane 6) and bottom strands (Fig. 3C, lane 6), indicating a preference, in the binary complex, for an orientation opposite to that seen in the ternary complex. Because TFB makes critical contacts with residues in the TBP stirrup, mutant cACA, with its C-terminal stirrup disabled for TFB assembly, must have relied on its N-terminal half (the N-terminal stirrup and helix H1) to assemble TFB, and must have been forced by the latter to bind DNA in the opposite orientation (Fig. 3D).

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

Fig. 3

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(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, nACOPA and cACOPA 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 nACOPA and cACOPA 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.

No specific DNA cleavage was observed using a wild type TBP that had been alkylated with IAAOP, in parallel with mutants nACA and cACA (lanes 2-3 in Figs 3B and 3C), indicating that the observed cleavage patterns resulted from the site-specific coupling of OP to the cysteine residues introduced into the TBP stirrups.

This ternary complex geometry is not specific to the Pfu gdh promoter (Fig. 3A), for an identical TBP orientation was observed in ternary complexes assembled on the Methanococcus vannielii (Mva) tRNAval promoter, another well-characterized, strong archaeal promoter (Hausner et al., 1991) (Fig. S2, Supporting information). The affinity cleavage patterns produced on these T/A-boxes were similar, suggesting that ternary complexes at both promoters possess equivalent architectures. Taken together, these data demonstrate the ability of Mja TBP to assemble TFB using either of its subdomains, thus attaching a functional significance to the unusually high degree of symmetry exhibited by its two halves, especially the absolute conservation of its stirrups.

In view of the orientational symmetry of Mja TBP, one might wonder whether the gdh promoter's BRE still enforces unidirectional recruitment of RNAP. To answer this question, we inserted an initiator element on the BRE side of the T/A box with the identical spacing of the gdh transcription unit and checked for run-off transcription in the upstream (anti-gdh) direction. None could be detected, either with wild type or cAAA TBP with its disabled C-terminal stirrup (Fig. 4, lanes 3 and 4, respectively). We conclude that only the strong gdh BRE upstream of the T/A box is functional, and that it enforces a unique orientation of TFB which, in turn, enforces a unique orientation of transcription.

Fig. 4. Functional dominance of the gdh promoter BRE.

Fig. 4

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(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 Pgdh 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.

TBP stirrup symmetry, TBP-TFB interaction and activated transcription

The correlation of activation of RNA pol II transcription in vivo with TBP–TFIIB interaction has been analyzed extensively, and has been noted to vary widely. In yeast, to a large extent, the TBP–TFIIB interaction appears dispensable for transcriptional activation by various acidic activators, including VP16 (Chou & Struhl, 1997, Lee & Struhl, 1997). A more variable pattern of dependency on the TBP–TFIIB interaction was observed in HeLa cells, in which VP16-activated transcription was found to be entirely dependent on strong TBP–TFIIB contacts, whereas SP1-mediated transcription showed little requirement for it (Tansey & Herr, 1997).

The next experiment examines the relationship of Mja TBP stirrup symmetry and the observed redundancy in TBP-TFB contacts in transcription that is activated by the positive regulator Mja Ptr2. Ptr2 is a homologue of the Lrp/AsnC family of bacterial transcription regulators that are among the most widely disseminated archaeal DNA-binding proteins, and has been shown to activate transcription by its cognate core transcription apparatus in vitro (Ouhammouch et al., 2003, Ouhammouch et al., 2004). Transcriptional activation by Ptr2 is generated from promoter-proximal upstream activating DNA sites (UAS), at least in part, by recruiting TBP to the promoter (Ouhammouch et al., 2003). We tested ability of Mja transcription driven by TBP mutants nAAA and cAAA, as well as the corresponding cysteine variants nACA and cACA, to respond to activation by Ptr2 at the previously described rb2 promoter construct with an optimum Ptr2 UAS (Ouhammouch et al., 2005). As shown in Fig. 5A, rb2 transcription specified by TBP mutants nAAA (lanes 4-6) and cAAA (lanes 7-9) failed to respond to Ptr2, in contrast to wild type TBP (lanes 1-3); the corresponding cysteine variants nACA and cACA exhibited the same failure of activation by Ptr2 (Fig. S3, Supporting information). In contrast, when assayed on the strong gdh promoter, TBP nAAA supported nearly wild type levels of basal transcription, and cAAA supported ∼ 45% of wild type-level activity (Fig. 2). As shown in Fig. 5B (lanes 10-12), increasing TBP nAAA concentrations (up to 160 nM; the optimum wild type TBP concentration being ∼ 20 nM) did not significantly affect its level of response to Ptr2. Hydroxyl radical footprinting experiments showed that nAAA TBP exhibited only a mild DNA-binding defect on its own, and that this defect was partially relieved by TFB (results not shown).

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

Fig. 5

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(A) Prb2 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 Prb2 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.

Taken together, these results point to an essential functional role of both TBP stirrups in Ptr2-activated transcription. The implications of this finding are discussed below.

Discussion

The TATA-binding proteins (TBPs) of contemporary archaea and eukaryotes are the products of ancestral gene duplication and fusion events resulting in a protein with pseudo-two-fold symmetry. TBP initiates the assembly of archaeal promoter complexes and many eukaryotic pol II complexes by binding to highly similar core DNA sites, the T/A and TATA boxes, respectively. There is insufficient asymmetry in the TATA box and its interaction with the DNA-binding surface of TBP to ensure completely unidirectional assembly. That this was not more quickly recognized is primarily due to a historical accident: the crystal structures of Arabidopsis and yeast TBP-TATA box complexes showed a unique orientation of TBP on DNA with the upstream-lying half of the TATA box bound by the C-terminal half of TBP (Kim et al., 1993a, Kim et al., 1993b, Kim & Burley, 1994), and the subsequently solved structures of TFIIB/TBP/TATA box and TFIIA/TBP/TATA box complexes showed the same orientation (Nikolov et al., 1995, Geiger et al., 1996). In fact, early experiments had already suggested that, for RNA pol II, a TATA box could not be a sufficient determinant for unique transcriptional polarity (Xu et al., 1991), and ambidirectional binding of TBP to TATA boxes in solution was eventually demonstrated directly (Cox et al., 1997).

That conflict crystallized (as it were) when an archaeal TFB-TBP complex was found to be oriented in the opposite direction on a T/A box (Kosa et al., 1997). The resolution came with the discovery of the pol II promoter TFIIB recognition element (BRE) and its archaeal counterpart, located just upstream of the TATA box (Lagrange et al., 1998, Qureshi & Jackson, 1998), followed by the determination of the “correctly” oriented structure of an archaeal TFB-TBP complex on BRE-containing DNA (Littlefield et al., 1999).

Methanocaldococcus jannaschii (Mja) TBP and its counterparts from closely related methanococcales, Methanothermococcus thermolithotrophicus (Mth) and Methanococcus maripaludis (Mma) are highly homologous (81.3% and 74.7% identity, respectively). A comparative analysis of their ability to respond to transcriptional activation by Mja Ptr2 uncovered the surprising finding that Mja TBP is a preferred partner (relative to the conjugate Mth and Mma TBPs) for Ptr2-mediated transcriptional activation (Ouhammouch & Geiduschek, 2005). A mutational analysis of Mja TBP to understand the basis for this preference in turn led to the experiments that are presented here.

As already mentioned, the ability of TFIIB and TFB to dictate the orientation of TBP on the TATA or T/A box, respectively, is conferred by protein-protein contacts that appose their C-terminal cyclin repeat with helix H′1 and the loop of the C-terminal half of TBP (C-terminal stirrup). The similarity of the N- and C-halves of archaeal TBPs is especially pronounced in the Methanococcales and Thermoplasmatales, as already mentioned, including complete conservation of the N- and C-terminal stirrups (Fig. 1C). Incidentally, in the Mja protein, the high degree of similarity between the N- and C-terminal halves extends to helices H1 and H′1. In that sense, these TBPs retain more of the presumptive ancestral TBP symmetry, in fact, more so than the TBP of the newly sequenced deeply branching korarchaeote Candidatus Korarchaeum cryptofilum, whose genome appears to be a composite of more euryarchaea- and crenarchaea-like parts (Elkins et al., 2008).

The functional consequences of this symmetry for assembly of pre-initiation TFB/TBP/promoter complexes on our selected promoters are remarkable. Although single alanine substitutions in the C-terminal stirrup of yeast and human TBP corresponding to Mja TBP residues E126, E128 and Q129 impair assembly of TFIIB-TBP complexes ∼10- to 100-fold in the case of the yeast proteins (Lee & Struhl, 1997) and ∼15- to 25-fold for the human counterparts (Tang et al., 1996), we find that the triple-alanine mutant at the corresponding positions in Mja TBP retains nearly half of its transcriptional activity at three strong promoters: the Pfu gdh promoter (Fig. 2), and two versions of the Mja ptr1 promoter (the wild type and a variant containing up-mutations in the BRE; data not shown). The corresponding triple mutant in the N-terminal stirrup, on the other hand, did not exert a significant effect on gdh transcription, but abolished activity in combination with the C-terminal stirrup mutations (ncAAA; Fig. 2). These findings suggested the possibility that Mja TFB assembly on the BRE is compatible with either orientation of Mja TBP on the T/A box. That this is the case was confirmed by direct footprinting with Cu-phenanthroline-derivatized TBP (Figs 3 and S2, Supporting information). At the same time, forcing the TFB interaction with the N-terminal stirrup of TBP (in mutant cAAA) did not affect the polarity of RNAP recruitment and transcription (Fig. 4). Thus, Mja TBP is literally ambidextrous with regard to basal transcription.

The situation is different for Ptr2-activated transcription. Here, mutagenizing the N-terminal downstream-facing stirrup, which has no effect on basal transcription at strong promoters (Fig. 2 and results not shown), nearly completely eliminates activation by Ptr2 (Fig. 5). So does mutating the C-terminal stirrup (Fig. 5), although that does also reduce basal transcription at strong promoters (Fig. 2 and results not shown). At first glance, these findings might be taken to indicate that both stirrups of TBP interact with Ptr2 and/or that they are required to mediate activation by Ptr2. The other experiments presented here suggest two possible, and we think more probable, scenarios. In the first, the downstream-facing stirrup, which is not engaged with TFB does indeed mediate activation by Ptr2 and the three alanine substitutions in TBP nAAA essentially eliminate this interaction. The residual transcriptional activity of mutant cAAA requires turning TBP around on the T/A box so that its mutated C-terminal stirrup also faces downstream (Fig. 3D). As a consequence, Ptr2 “sees” the same defect in both nAAA and cAAA. Alternatively, Ptr2 could be the factor determining the orientation of TBP on the T/A box. Ptr2 recruits both TBP mutants nAAA and cAAA to DNA through interaction with their respective wild type stirrups (facing the downstream edge of the T/A box). As a result, the mutated stirrup of either TBP mutant is made to face upstream, thereby preventing TFB from entering the promoter complex. Each scenario would result in a failure of activation by Ptr2. Experiments aimed at distinguishing these two alternatives and delineating the network of interactions within the Ptr2/TBP/TFB/DNA complex are currently underway.

Experimental procedures

Proteins

Methanocaldococcus jannaschii (Mja) RNAP was prepared as described previously (Ouhammouch et al., 2003). Mja TBP and TFB were overproduced in E. coli and purified as described (Ouhammouch et al., 2003). Genes encoding the TBP mutants listed in Fig. 1B (nAAA: E35A-E37A-Q38A; cAAA: E126A-E128A-Q129A; ncAAA: E35A-E37A-Q38A-E126A-E128A-Q129A; nACA: E35A-E37C-Q38A; cACA: E126A-E128C-Q129A; ncACA: E35A-E37C-Q38A-E126A-E128C-Q129A) were generated by the Kunkel method of oligonucleotide-directed mutagenesis (McClary et al., 1989, Kunkel et al., 1991). Overproduction and purification followed the procedure described for wild type TBP (Ouhammouch et al., 2003).

TBP derivatization with 5-iodoacetamido-1,10-phenanthroline

Wild type, nACA and cACA TBPs were chromatographed individually, twice, through a P-30 Micro Bio-Spin column (BioRad) equilibrated with conjugation buffer [50 mM Na-Hepes, pH 7.8, 300 mM NaCl, 0.1 mM EDTA, 10 mM TCEP, 5% (w/v) glycerol]. To 100 μl volume of 50 μM solutions of buffer-exchanged proteins were added 2 μl of 100 mM 5-iodoacetamido-1,10-phenanthroline (IAAOP) in dimethylformamide, and allowed to react in the dark for 3 h at room temperature, and for an additional 15 h at 4°C. Dimethylformamide and excess IAAOP were subsequently removed by gel filtration through two P-30 Micro Bio-Spin columns equilibrated with storage buffer [50 mM Na-Hepes, pH 7.8, 300 mM NaCl, 0.1 mM EDTA, 25% (w/v) glycerol], and the concentrations of the derivatized proteins were checked by SDS-PAGE.

DNA

The 75-bp Pyrococcus furiosus (Pfu) gdh promoter probes used in DNA affinity cleavage experiments were generated by annealing oligonucleotides ON523 (5′-CCCAAAAGGATTTCCACTCTTGTTTACCGAAAGCTTTATATAGGCTATTGCCCAAAAATGTATCGCCAATCACCC-3′) and ON524 (5′-GGGTGATTGGCGATACATTTTTGGGCAATAGCCTATATAAAGCTTTCGGTAAACAAGAGTGGAAATCCTTTTGGG-3′). The 75-bp Mva tRNAval promoter probes were generated by annealing oligonucleotides ON525 (5′-GGGCAGTACTCCGACTCTAGAGGATCCAAAAAGTTTATATATCATGAATACTATGTTTAGTTTGCTCTCAGTGGG-3′) and ON526 (5′-CCCACTGAGAGCAAACTAAACATAGTATTCATGATATATAAACTTTTTGGATCCTCTAGAGTCGGAGTACTGCCC-3′).

The 162-bp Pfu gdh transcription template was generated by PCR using oligonucleotides ON538 (5′-GCGCCTAATCAAATAAACAAAAGGATTTCCAC-3′) and ON539: (5′-GGGCAGCTCTTTCAAGTTGCTTAATAACA-3′), and extends from bp −79 to +83 (relative to the transcriptional start as +1). The 203-bp mut-gdh transcription template was generated by mutagenic PCR using oligonucleotides ON579 (5′-GCCTGAGGAACACCTTTATATTTTTGA-3′) and ON539 as end primers, and oligonucleotide ON580 (5′-GCTTTCGGTAAACAAGAGTGTCGCTCCTTTTGTTTATTTGATTAGG-3′) and its reverse-complement (ON581) as mutagenic primers. This template extends from bp −120 to +83. Ptr2-activated transcription was analyzed using the previously characterized derivative of the Mja rb2 promoter with both Ptr2-binding sites of its UAS converted to the SELEX-derived consensus (5′-GGACGATTTTCGTCC-3′) and a third Ptr2-binding site (Site X) centered at bp +7 (relative to the transcriptional start as +1) mutated. This is the promoter construct designated as cons:cons:mut in Fig. 5 of (Ouhammouch et al., 2005). Complete DNA sequences are available on request.

In vitro transcription

For basal transcription, reaction mixtures were assembled in 50 μl (final volume) of transcription buffer [20 mM Na-Hepes, pH 7.8, 10 mM MgCl2, 250 mM NaCl, 1 mM DTT, 2.5% (w/v) glycerol]. Preinitiation complexes were assembled with 4 nM linear template DNA (gdh promoter; 200 fmol), 40 nM TFB, ∼30 nM purified Mja RNA polymerase and the indicated concentrations of TBP, for 20 min at 65°C. Single round transcription was then initiated at the same temperature by the addition of ATP, CTP, and GTP to 0.4 mM each, [α-32P]UTP [3,000 Ci (1 Ci = 37 GBq)/mmol] to 32 μM, poly(dI-dC):poly(dI-dC) to 80 μg/ml, and 18 units of RNase inhibitor, for 20 min. The addition of 80 μg/ml poly(dI-dC):poly(dI-dC) prevents reinitiation, thus limiting transcription to a single round. Ptr2-activated transcription followed the previously described procedure (Ouhammouch et al., 2003). Sample preparation (extraction with phenol/chloroform/isoamyl alcohol, and precipitation with ethanol), resolution by electrophoresis on denaturing 5% polyacrylamide gel, visualization by phosphor imaging, and quantification followed standard procedures.

DNA affinity cleavage

Protein–DNA complexes were assembled in 100 μl of binding buffer (20 mM Na-Hepes, pH 7.8, 10 mM MgCl2, 250 mM NaCl) with 400 fmol of 5′-end-32P-labeled DNA (4 nM), 120 nM TFB and 120 nM TBP-OP. Reaction mixtures were incubated at 65°C for 15 min and were subjected to DNA cleavage at the same temperature by adding CuSO4 to 30 μM, mercaptopropionic acid to 2.6 mM and H2O2 to 0.5 mM. Reactions were quenched after 8 min by addition of 50 μl of stop-mix [7.5 mM Tris-HCl, pH 8.0, 0.75 mM EDTA, 9 mM neocuproin, 2.8% (w/v) glycerol, 0.2% (w/v) SDS]. After extraction with phenol/chloroform/isoamyl alcohol and precipitation with ethanol, DNA cleavage products were resolved by electrophoresis on denaturing 10% polyacrylamide gels and visualized by phosphor imaging. The intensity of cleavage in this assay (Figs 3 and S2, Supporting information) is the product of binding affinity and precise orientation of the Cu-OP moiety within the DNA minor groove.

Acknowledgements

We thank G.A. Kassavetis for helpful discussions and advice, and the NIGMS for support of this research.

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