A Role for the Conserved GAFTGA Motif of AAA+ Transcription Activators in Sensing Promoter DNA Conformation^*

Gene transcription in bacteria is catalyzed by the multisub-unit DNA-dependent RNA polymerase (RNAP).⁵ The catalytically competent core form of bacterial RNAP is a five-subunit enzyme (α₂ββ′ω; E), which has to associate with a sixth subunit, the σ factor, for promoter-specific and regulated initiation of gene transcription. On the basis of differences in mechanism of action and amino acid sequence, bacterial σ factors are classified into two families. Most bacterial σ factors belong to the σ⁷⁰ family, named after the prototypical housekeeping σ factor, σ⁷⁰,of Escherichia coli. The major variant bacterial σ factor belongs to the σ⁵⁴ class.

Transcription initiation by RNAP containing σ⁷⁰ (Eσ⁷⁰) and σ⁵⁴ (Eσ⁵⁴) is mechanistically distinct. Eσ⁷⁰ recognizes promoters that contain consensus sequences centered at DNA positions -35 and -10, respectively, from the transcription start site (at +1). The initial transcriptionally inactive Eσ⁷⁰·DNA complex, called the closed complex, can spontaneously isomerize to form the transcriptionally active open complex, in which the DNA strands are separated and the RNAP is poised for RNA synthesis. In contrast, Eσ⁵⁴ forms closed complexes on promoters that contain consensus sequences centered at DNA positions -24 and -12 (1). Closed complexes formed by Eσ⁵⁴ remain inactive for transcription unless activated by a specialized type of transcription activator protein that belongs to the AAA (ATPases associated with various cellular activities) protein family (2). Eσ⁵⁴-dependent transcription activators (from now on referred to as AAA activators) bind to DNA sites located (∼150–200 bases) upstream of the promoter (known as upstream activating sequences) and use the energy derived from ATP binding and hydrolysis to remodel the Eσ⁵⁴ closed complex (2). The ATP hydrolysis-dependent binding interactions between the AAA activator and Eσ⁵⁴ closed complex trigger a series of protein and DNA isomerization events in the Eσ⁵⁴ closed complex, which result in the formation of the open complex. The major energetically favorable binding site for the AAA activator within the Eσ⁵⁴ closed complex is the N-terminal Region I domain of σ⁵⁴ (see Fig. 1A) (3), which, in the closed complex, is located at the -12 consensus promoter region, where DNA opening for open complex formation nucleates (4). At the -12 promoter region, σ⁵⁴ Region I meditates tight binding to a repressive fork junction structure and so prevents open complex formation in the absence of activation. Region I of σ⁵⁴ is associated with a range of properties of Eσ⁵⁴ (1). These include maintaining the closed complex transcriptionally silent prior to activation (5), stabilizing the open complex once it is formed (6), and conformational signaling to a structurally conserved DNA-interacting domain(s) of the catalytic β′ subunit of RNAP (7) required for stable open complex formation. Region I ofσ⁵⁴ has been shown to make extensive interactions with the catalytic β and β′ subunits of RNAP (8).

The AAA activators of Eσ⁵⁴ are mechanochemical P-loop ATPases of the AAA family (2, 9) and can use ATP hydrolysis to remodel the Eσ⁵⁴ closed complex to trigger open complex formation. Structural analysis of one model AAA activator, E. coli PspF (phage shock protein F) (10), suggests that mobile loops, called loops 1 (L1) and 2 (L2), within a hexameric assembly contact Region I of σ⁵⁴ (see Fig. 1, B and C) and that these contacts form during the energy-coupling process that transmits a conformational change around the γ-phosphate of the ATP-binding site on PspF to changes in the Eσ⁵⁴ closed complex conformation, which subsequently triggers open complex formation. Specifically, an invariant threonine residue in L1 (Thr⁸⁶ in PspF) (see Fig. 1C) is thought to engage with σ⁵⁴ Region I during the energy-coupling process (10, 11). The ATP hydrolysis-dependent movements of L1 and L2 have also been observed in the AAA activator Aquifex aeolicus NtrC1 (nitrogen regulatory protein C1) (12).

Current models for energy coupling by PspF indicate that a range of functional states of the AAA domain of PspF will exist, depending upon its nucleotide-bound state, and that more than one conformation of σ⁵⁴ Region I will exist for open complex formation to occur (10). To elucidate the role of σ⁵⁴ Region I and the invariant Thr⁸⁶ residue in the energy-coupling process, we have sought altered forms of σ⁵⁴ that can compensate for defects displayed by a partially functional PspF variant containing a serine substitution at Thr⁸⁶ in L1 (3, 11). In a previous study, we established that the T86S form of L1 of PspF is defective in interaction with the Eσ⁵⁴ closed complex in the presence of the ATP hydrolysis transition state analog ADP·AlF_x (11). The ADP·AlF_x-dependent complex between PspF and the Eσ⁵⁴ closed complex (referred to as the ternary complex) is regarded as an intermediate state that is established en route to full open complex formation (3, 13). Here, we describe the isolation of a new mutant form of σ⁵⁴, σ⁵⁴(G4L) (see Fig. 1A), capable of suppressing the defects of PspF(T86S) evident in ternary complex formation with the Eσ⁵⁴ closed complex and during in vitro transcription initiation. We show that the “to-be-melted DNA” downstream of the -12 consensus promoter region somehow negatively affects the ability of PspF(T86S) to stably interact with the Eσ⁵⁴ closed complex. The properties of PspF(T86S) andσ⁵⁴(G4L) imply a role for the conserved GAFTGA motif in PspF and, by extension, in other AAA activators of Eσ⁵⁴ in “sensing” the conformation adopted by the DNA downstream of the -12 promoter region and coupling this information to Eσ⁵⁴ via Region I to allow open complex formation.

EXPERIMENTAL PROCEDURES

Design and Construction of a σ⁵⁴ Region I Fragment 2–14 Mutant Library—Klebsiella pneumoniae σ⁵⁴ Region I amino acids 2–14 were subjected to random mutagenesis using a novel combinatorial codon-based mutagenesis approach that enabled the mutagenic rate to be controlled to obtain σ⁵⁴ variants with few amino acids substitutions (14). A detailed background of this mutagenesis method is described by Gaytán et al. (14). This method is based on the use of five dinucleotides designated B₂B₃ (AA, TT, AT, GC, and CG). Mutant codons are assembled by tandem addition of a diluted mixture of the five dinucleotides to the growing oligonucleotide and a mixture of four nucleotides to generate 20 trinucleotides that encode a set of 18 amino acids. Thus, in this method, wild-type codons are doped with a set of mutant codons during oligonucleotide synthesis to generate random combinatorial libraries of primers that contain few codon replacements per variant. The mutagenic rate is controlled by varying the percentage of B₂B₃ dimers used to “dope” the second and third positions of the wild-type codons targeted for mutagenesis. An oligonucleotide, called oligonucleotide A (72-mer), with sequence 5′-tattggccctgcatATG-AAG/B₁B₂B₃-CAA/B₁B₂B₃-GGT/B₁B₂B₃-TTG/B₁B₂B₃-CAA/B₁B₂B₃-TTA/B₁B₂B₃-AGG/B₁B₂B₃-CTA/B₁B₂B₃-AGC/B₁B₂B₃-CAA/B₁B₂B₃-CAG/B₁B₂B₃-CTT/B₁B₂B₃-GCC/B₁B₂B₃-atgacgccacaactg-3′ was assembled. The wild-type σ⁵⁴ codons subjected to mutagenesis are in uppercase letters. A second oligonucleotide, called oligo-nucleotide B (63-mer), with sequence 5′-ttgctggagttctagagtggacagctgcagtagacgaatcgcctgctgcagttgtggcgtcat-3′ was designed to complement the 3′-flanking arm of oligonucleotide A. The complementary sequences of both oligonucleotides are shown in boldface. The flanking arms included two restriction sites, NdeI and XbaI (underlined). The introduction of the restriction sites did not change the wild-type coding sequence of σ⁵⁴. The duplex DNA library of the target region was generated by the extension of complementary oligonucleotide B over mutagenic oligonucleotide A using the Klenow fragment (3′ → 5′ exo^-) of DNA polymerase I. Briefly, an equimolar concentration of oligonucleotides A and B (500 pmol) was mixed in Klenow buffer (50 mm Tris-HCl (pH 7.2 at 25 °C), 10 mm MgSo₄, and 1 m m DTT) containing all four dNTPs. For the annealing step, the mixture was heated at 70 °C for 15 min and allowed to cool to room temperature. 1 μl of Klenow fragment (5 units/μl) was added, and the reaction was incubated at 37 °C for 1 h. Following this, the Klenow fragment was heat-inactivated at 70 °C for 15 min. The duplex DNA library was separated by gel electrophoresis and purified from single primer species on a 2% (w/v) agarose gel using a Qiagen gel extraction kit following the manufacturer's instructions. The duplex DNA library was digested with NdeI and XbaI and used to replace the wild-type σ⁵⁴ residues corresponding to positions 2–14 in pVB009 (15). The in vivo screening for the rpoN clones capable of recovering the activation defect of NifA(T308S) (nitrogen fixation protein A) was performed as described previously (3). Briefly, the mutant library in pVB009 (ampicillin resistance) was then transformed into E. coli strain (TH1 (ΔrpoN)) and selected on X-gal (20 μg/μl) containing nitrogen-free medium.

In Vivo Activity Assays—The in vivo β-galactosidase assays were conducted as described previously (3, 15) in E. coli strain TH1 (ΔrpoN) containing plasmids pRT22 (chloramphenicol resistance) and pWKS130 (kanamycin resistance). pRT22 is a reporter plasmid and contains the σ⁵⁴-dependent K. pneumoniae nifH promoter fused to the lacZ gene. pWKS130 carries the three different Bradyrhizobium japonicum nifA alleles (wild-type nifA, nifA(T308S), and nifA(T308V)) used in this study. The clones that displayed the desired phenotype were sequenced to establish that mutation(s) were located only within amino acids 2–14 of σ⁵⁴.

Site-directed Mutagenesis—σ⁵⁴ and PspF-(1–275) containing single amino acid substitutions were constructed using the Stratagene QuikChange mutagenesis kit following the manufacturer's instructions. The templates used for the σ⁵⁴ and PspF-(1–275) mutagenesis reaction were pVB009 and pPB1 (16), respectively.

Proteins—For protein overproduction, NdeI-HindIII fragments containing wild-type and mutant (G4L) rpoN genes were cloned into pET28b⁺ (Novagen) and overproduced and purified as described previously (17). Wild-type and mutant variants of E. coli PspF-(1–275) were constructed, overproduced, and purified exactly as described (16, 17). Wild-type E. coli core RNAP was purchased from Epicentre Technologies (Madison, WI). The concentrations of all protein preparations were determined using a Bio-Rad protein assay kit accordingly to the manufacturer's instruction. To further ascertain that equal quantities of proteins were used in the assays, each preparation was checked by SDS-PAGE analysis.

Promoter DNA Probes and Proteins—The Sinorhizobium meliloti nifH homoduplex and heteroduplex promoter probes used in this study were constructed and ³²P-5′-end-labeled exactly as described previously (17).

Native Gel Mobility Shift Assays—These were performed exactly as described previously (17). The gels were analyzed, and the complexes were quantified using a Fuji FLA-5000 fluorescent image analyzer. All native gel mobility assays were repeated at least twice, and the values shown in the figures represent an average of these replicates with an error range of ±6%.

In Vitro Transcription Assays—These were performed exactly as described previously (7), but with a range of incubation times (specified in the figure legends). The transcription reactions were repeated at least twice, and the values shown in the figures represent an average of these replicates with an error range of ±5%.

RESULTS

In Vivo Isolation and Characterization of σ⁵⁴ Mutants That Recover the Activity of the Activation-defective T308S Mutation in the AAA Activator NifA—Initially, we screened in vivo for altered forms of K. pneumoniae σ⁵⁴ that were able to recover the activity of the AAA activator B. japonicum NifA containing the T308S substitution in L1 (18). The B. japonicum NifA(T308S) protein is drastically impaired only in transcription activation. Neither ATP hydrolysis nor oligomerization functions are affected by the T308S mutation, indicating a defect in coupling ATP hydrolysis to open complex formation (18). We used B. japonicum NifA instead of E. coli PspF for the in vivo experiments because in vivo assay systems for measuring activation of transcription are well established and documented for the NifA protein (3, 18). Previous results indicated that σ⁵⁴ Region I amino acids 6–14 are important for binding to the AAA activator (11). Thus, we subjected σ⁵⁴ amino acids 2–14 to a codon-based mutagenesis approach (14) (see “Experimental Procedures”). The mutant σ⁵⁴ library was transformed into E. coli strain TH1, which has a deletion of the rpoN gene, encoding σ⁵⁴, and contains plasmids pRT22 (3) and pWKS130 (18). pRT22 is a reporter plasmid and contains the σ⁵⁴-dependent K. pneumoniae nifH promoter fused to the lacZ gene. pWKS130 constitutively encodes the NifA(T308S) activator protein. The transformed E. coli TH1 cells were plated onto X-gal-containing agar, and blue-white screening was used to identify σ⁵⁴ mutants capable of recovering NifA(T308S) activity. In this experimental system, E. coli cells containing σ⁵⁴ mutants capable of recovering the NifA(T308S) activity will produce colonies displaying a deep blue color. We isolated six colonies (rpoN-sup1–6) that displayed a deep blue color. An in vivo β-galactosidase activity assay was used to quantitatively assess the ability of these six mutant clones (rpoN-sup1–6) to recover the activity of the NifA(T308S) protein. As shown in Table 1, the NifA(T308S)-dependent expression of lacZ was ∼6–20-fold higher in the presence of the six mutant σ⁵⁴ clones than in the presence of wild-type σ⁵⁴. However, we noted that the level of transcriptional activity of the rpoN-sup clones with NifA(T308S) represented ∼15–20% of the transcriptional activity obtained with wild-type NifA. Thus, this result suggests that all six mutant σ⁵⁴ clones, at least in the context of this assay, are able to specifically recover the activity of NifA(T308S).

Sequence analyses of clones rpoN-sup1–6 revealed that all, except clone rpoN-sup4, contained three to five amino acid substitutions that were widely distributed within the σ⁵⁴ Region I segment (amino acids 2–14) that was subjected to mutagenesis. Analysis of randomly chosen clones from the library that did not display a deep blue color also revealed multiple amino acid substitutions within this σ⁵⁴ Region I segment. In line with the key objective of this study, we did obtain six mutant rpoN clones capable of suppressing the activation defect displayed by the NifA(T308S) protein, but failed to obtain rpoN clones with single amino acid substitutions.

Interestingly, a common feature in all mutant clones that rescued NifA(T308S) was the high occurrence of hydrophobic amino acids at the mutated positions. Notably, all mutant clones had a mutation of the non-conserved glycine codon at position 4 (Gly⁴), and in most cases (rpoN-sup2 and rpoN-sup4–6), Gly⁴ was changed to a hydrophobic residue (Table 1). In most rpoN genes sequenced so far, position 4 is represented by a hydrophilic residue. From Table 1, it seems that the substitution of the conformationally flexible glycine residue at position 4 of K. pneumoniae σ⁵⁴ Region I is a requirement for recovering the activity of NifA(T308S). Because, unlike the other clones, clone rpoN-sup4 contained substitutions only at Gly⁴ and Gln¹¹ and displayed the second highest activity in the β-galactosidase assays (Table 1), we separated the G4L/Q11F mutations to further investigate the role of G4L and Q11F substitutions in recovering the activity of NifA(T308S). Initially, we used the in vivo β-galactosidase activity assay (as described above) to establish whether the single mutants σ⁵⁴(G4L) and σ⁵⁴(Q11F) are able to express lacZ in the presence of wild-type NifA. As shown in Table 2, σ⁵⁴(G4L) was ∼20% more active than wild-type σ⁵⁴. In contrast, σ⁵⁴(Q11F) was ∼3–4-fold less active than either σ⁵⁴(G4L) or wild-type σ⁵⁴. Notably, the double mutant σ⁵⁴(G4L/Q11F) was ∼20% more active than wild-type σ⁵⁴ in expressing lacZ in a wild-type NifA-dependent manner. E. coli TH1 cell extracts were prepared and probed with anti-σ⁵⁴ antibodies to confirm that the stability of σ⁵⁴ was not affected by the Q11F mutation. The results showed that wild-type σ⁵⁴, σ⁵⁴(G4L), σ⁵⁴(Q11F), and σ⁵⁴(G4L/Q11F) were present at equal amounts under the assay conditions (data not shown). Thus, it appears that the Q11F single substitution in σ⁵⁴ is unfavorable for wild-type NifA-dependent activation of transcription, but that σ⁵⁴(Q11F) is expressed as a stable protein.

Next, we investigated whether the single mutants σ⁵⁴(G4L) and σ⁵⁴(Q11F) are able to recover NifA(T308S) activity as effectively as σ⁵⁴(G4L/Q11F). As expected, wild-type σ⁵⁴ was unable to express lacZ when NifA(T308S) was used for activation. Notably, only σ⁵⁴(G4L) was able to recover the activity of NifA(T308S) and expressed lacZ at an ∼6-fold higher level compared with wild-type σ⁵⁴ (Table 2). σ⁵⁴(Q11F) was unable to recover NifA(T308S) activity (Table 2). Thus, it seems that the G4L substitution is the significant mutation in σ⁵⁴ Region I that allows recovery of NifA(T308S) activity. However, the level of lacZ expression by σ⁵⁴(G4L) was ∼3-fold reduced compared with σ⁵⁴(G4L/Q11F)_. Thus, it seems that, in the context of σ⁵⁴(G4L/Q11F), the hydrophobic substitution at Gln¹¹ facilitates an improved interaction between Region I of σ⁵⁴ and L1 of NifA(T308S).

We assessed whether the σ⁵⁴ mutants are able to suppress the NifA(T308S) mutant in vivo in an allele-specific manner by using another NifA mutant carrying a different substitution at the same L1 position (NifA(T308V)). Like NifA(T308S), this mutant is impaired in transcription activation, but contains an amino acid at position 308 that is geometrically and spatially very similar to a threonine residue. The in vivo activation assay showed that neither σ⁵⁴(G4L) nor σ⁵⁴(G4L/Q11F) was able to recover the activity of the activation-defective NifA variant containing the T308V substitution (Table 2). Hence, it appears that σ⁵⁴(G4L) specifically recovers NifA(T308S) activity.

Eσ⁵⁴(G4L) Recovers the Activation-defective Property of PspF(T86S)-(1–275) in an in Vitro Transcription Assay—The in vivo analysis revealed that a single amino acid substitution at position 4 (G4L) in Region I of K. pneumoniae σ⁵⁴ is sufficient to specifically recover the activity of NifA(T308S) and to explain the molecular basis for the activation defect of the NifA(T308S) mutant. To determine the properties of σ⁵⁴(G4L) responsible for recovering the activity of NifA(T308S), we conducted a series of in vitro experiments that measured activator-dependent output by Eσ⁵⁴ at several steps leading to productive transcription initiation. For the in vitro assays, we used the AAA domain of the AAA activator E. coli PspF (PspF-(1–275)) (11) containing the T86S substitution (the equivalent of the T308S mutation in NifA) because, in contrast to the NifA protein, (i) PspF-(1–275) is far better suited for in vitro studies and (ii) well established experimental assays exist to study PspF-(1–275)-dependent activation of Eσ⁵⁴ transcription (3, 17). Furthermore, PspF-(1–275) lacks the domain containing the helix-turn-helix motif and is thus able to efficiently activate transcription (in vivo and in vitro) from solution without the need to bind to upstream activating sequences, a property that significantly simplifies in vitro experimental design (19).

σ⁵⁴ containing the G4L mutation was purified as an N-terminally hexahistidine-tagged fusion protein. Initially, we determined that σ⁵⁴(G4L) bound core RNAP as well as wild-type σ⁵⁴ (using a simple native gel assembly assay) and that its ability to initiate transcription strictly relied upon the AAA activator and ATP hydrolysis (data not shown; see below). We used a dinucleotide-primed abortive initiation transcription assay to recapitulate in vitro with PspF-(1–275) the changes in transcription activation properties measured in vivo with NifA. As indicated in the reaction schematic in Fig. 2, open complex formation was stimulated by exposing the Eσ⁵⁴ closed complex to wild-type PspF-(1–275) or PspF(T86S)-(1–275). Closed complexes were formed on an 88-bp-long linear DNA fragment containing the σ⁵⁴-dependent S. meliloti nifH promoter (Fig. 1D, native). Control gel shift experiments indicated that approximately equal amounts of closed complexes were formed by wild-type and mutant Eσ⁵⁴ under the experimental conditions (data not shown). Following activation, the promoter complexes were challenged with the polyanion heparin. Heparin is a commonly used DNA competitor that disrupts closed complexes or promoter complexes that have not isomerized properly and/or in which DNA opening is not stable. Thus, only transcriptionally proficient stable open complexes are relatively heparin-resistant. As shown in Fig. 2 (lanes 1 and 2), ∼90% less abortive transcripts were formed by wild-type Eσ⁵⁴ when open complex formation was stimulated by PspF(T86S)-(1–275) than by wild-type PspF-(1–275). In contrast, in reactions containing Eσ⁵⁴(G4L), equal amounts of abortive transcripts were formed (Fig. 2, lanes 3 and 4), regardless of whether wild-type PspF-(1–275) or PspF(T86S)-(1–275) was used to stimulate open complex formation. Similar results were obtained when the assay was done using a supercoiled plasmid harboring the S. meliloti nifH promoter in which the synthesis of full-length transcripts was measured (data not shown). Overall, these results clearly recapitulate the in vivo observations (see above) and show that the G4L substitution in Region I of σ⁵⁴ is able to recover, both in vivo and in vitro, the activation defect conferred by mutation of the invariant threonine residue in L1 of PspF and NifA in a transcription assay.

Mutation G4L in σ⁵⁴ Enables PspF(T86S)-(1–275) to Stably Interact with the Closed Complex in a Nucleotide-dependent Manner—Previously, we demonstrated that PspF(T86S)-(1–275) is unable to form the ternary complex with a binary σ⁵⁴·promoter DNA or Eσ⁵⁴·promoter DNA complex in the presence of the ATP hydrolysis transition state analog ADP·AlF_x (11). We now wished to determine whether PspF(T86S)-(1–275) can stably bind to S. meliloti nifH promoter complexes reconstituted with σ⁵⁴(G4L) and Eσ⁵⁴(G4L) in the presence of ADP·AlF_x. Promoter complexes were formed on a DNA probe that mimicked the conformation of the promoter DNA adopted within the closed complex. This promoter probe, referred to as the -12/-11 probe, contained a 2-base heteroduplex segment immediately downstream of the consensus GC sequence (Fig. 1D). As shown in Fig. 3 (A and B, compare lanes 5 and 6), PspF(T86S)-(1–275) was able to form the ternary complex with promoter complexes reconstituted with σ⁵⁴(G4L) or Eσ⁵⁴(G4L) as well as wild-type PspF-(1–275). Control assays established that ternary complex formation between σ⁵⁴(G4L) and PspF(T86S)-(1–275) was dependent on ADP·AlF_x (data not shown). As expected, PspF(T86S)-(1–275) did not form the ternary complex when promoter complexes were reconstituted with wild-type σ⁵⁴ or Eσ⁵⁴ (Fig. 3, A and B, lanes 3) (11).

We extended the ternary complex formation assay to determine whether the G4L mutation in σ⁵⁴ specifically recovers the activation defect of PspF(T86S)-(1–275). Four different PspF-(1–275) variants (PspF(T86A)-(1–275), PspF(F85A)-(1–275), PspF(F87L)-(1–275), and PspF(F85W)-(1–275)) that were defective in ternary complex formation with promoter complexes reconstituted with wild-type σ⁵⁴ (Fig. 3C, lanes 2–6) were used for this purpose to help determine the specific defects in PspF(T86S)-(1–275). σ⁵⁴(G4L) specifically formed the ternary complex only with PspF(T86S)-(1–275) (Fig. 3C, lanes 8–12). Identical results were obtained in experiments conducted in the presence of core RNAP (data not shown). Overall, the results suggest that the in vivo properties of σ⁵⁴(G4L) with NifA can be reproduced in vitro with PspF-(1–275). Furthermore, in full agreement with the in vivo data (Table 1), the results from the in vitro experiments suggest that the G4L substitution in Region I of σ⁵⁴ specifically allows the recovery of the activity of AAA activators containing only the Thr-to-Ser substitution in L1.

The G4L Substitution in σ⁵⁴ Region I Allows Remodeling of Eσ⁵⁴ by PspF(T86S)-(1–275)—The in vitro transcription assays (Fig. 2) suggest that the G4L substitution does not merely contribute to an improved binding interaction between σ⁵⁴ and the AAA activator in the presence of promoter DNA, but also allows the latter to couple ATP hydrolysis-dependent conformational changes in PspF-(1–275) to Eσ⁵⁴. We conducted the next set of experiments to directly investigate this. Previously, we described a simple native gel assembly assay to monitor ADP·AlF_x-dependent and PspF-(1–275)-induced remodeling of Eσ⁵⁴ (13). This assay measures the ability of PspF-(1–275) to confer upon Eσ⁵⁴ the ability to bind a mutant heteroduplex promoter probe, referred to as the -12/-11-mut promoter probe (Fig. 1D), in an ADP·AlF_x-dependent manner. The -12/-11-mut promoter probe differed from the -12/-11 promoter probe in that it contained a non-wild-type template strand sequence in the heteroduplex region (Fig. 1D). Eσ⁵⁴ (and σ⁵⁴) bound poorly to the -12/-11-mut promoter probe (Fig. 3D, lane 1). However, in the presence of PspF-(1–275) and ADP·AlF_x,Eσ⁵⁴ (and σ⁵⁴) formed a stable complex on the -12/-11-mut promoter probe (Fig. 3D, lane 2). In the presence of ADP·AlF_x, PspF(T86S)-(1–275) did not confer upon wild-type Eσ⁵⁴ the ability to bind the -12/-11-mut promoter probe (Fig. 3D, lane 3), revealing a marked defect in PspF(T86S)-(1–275). We wished to investigate whether the G4L substitution allows Eσ⁵⁴ to be remodeled by PspF(T86S)-(1–275) in the presence of ADP·AlF_x so that it can then bind to the -12/-11-mut promoter probe. As shown in Fig. 3D,Eσ⁵⁴(G4L) bound equally well to the -12/-11-mut promoter probe in reactions containing wild-type PspF-(1–275) (lane 5) and PspF(T86S)-(1–275) (lane 6) in an ADP·AlF_x-dependent manner. Thus, it appears that the G4L substitution in Region I of σ⁵⁴ not only simply facilitates an interaction between Eσ⁵⁴ and PspF(T86S)-(1–275), but also allows Eσ⁵⁴ to undergo PspF(T86S)-(1–275)-induced conformational changes in an ADP·AlF_x-dependent manner.

Recovery of the Transcription Activity of PspF(T86S)-(1–275) by σ⁵⁴(G4L) Is Dependent on Promoter DNA Structure—To determine the molecular basis by which the G4L substitution in σ⁵⁴ Region I recovers the activity of PspF(T86S)-(1–275), we repeated the abortive transcription initiation assay using a heteroduplex promoter DNA probe. This heteroduplex probe contained a heteroduplex segment between positions -10 and -1 with respect to the transcription start site at +1 and so stably mimicked the conformation of the promoter DNA in the open complex (Fig. 1D). Our aim was to investigate whether the T86S substitution negatively influences stable DNA opening by Eσ⁵⁴. As shown in Fig. 4 (lanes 2 and 4), the use of the -10/-1 probe did not allow PspF(T86S)-(1–275) to significantly overcome its activation defect, suggesting that the molecular basis for its activation defect involves steps prior to engagement of Eσ⁵⁴ with melted DNA. Interestingly, however, the recovery of PspF(T86S)-(1–275) activity by σ⁵⁴(G4L), as seen on the native promoter probe (Fig. 4, compare lanes 2 and 6), was not readily evident on the -10/-1 promoter probe. As shown in Fig. 4, regardless of the type of Eσ⁵⁴ used, ∼60–70% less abortive transcripts were synthesized when abortive initiation was stimulated by PspF(T86S)-(1–275) in reactions containing the -10/-1 promoter probe (compare lanes 4 and 8). We next investigated whether PspF(T86S)-(1–275) can form the ternary complex (in the presence of ADP·AlF_x) with Eσ⁵⁴ and Eσ⁵⁴(G4L) bound to the -10/-1 probe. As expected, PspF(T86S)-(1–275) was able to form ternary complexes in reactions containing only Eσ⁵⁴(G4L) (data not shown). Thus, it seems that (i) stably pre-opening the DNA does not allow PspF(T86S)-(1–275) to overcome its activation defect and that (ii) the conformation adopted by the melted out promoter DNA region has a potential role in the recovery of PspF(T86S)-(1–275) activity by σ⁵⁴(G4L) in a transcription assay (recall that recovery with homoduplex DNA was better than with the -10/-1 probe) (Fig. 2). However, the conformation of the promoter DNA does not seem to influence the ability of σ⁵⁴(G4L) to recover the activity of PspF(T86S)-(1–275) to form the ADP·AlF_x-dependent ternary complex.

PspF(T86S)-(1–275) Can Interact with Eσ⁵⁴ in the Absence of Promoter DNA—The results so far demonstrate that the G4L mutation in Region I of σ⁵⁴ allows the recovery of the defective property of PspF(T86S)-(1–275), but only in a restricted range of functional assays. Notably, experiments with heteroduplex DNA (Fig. 4) suggested that the conformation of the promoter DNA has a role in the ability of PspF(T86S)-(1–275) to be recovered by Eσ⁵⁴(G4L) in a transcription assay. To investigate the role of promoter DNA in the recovery process, we conducted a simple native gel assembly assay to measure the ADP·AlF_x-dependent interaction between Eσ⁵⁴ and PspF-(1–275) in the absence of promoter DNA. As shown in Fig. 5 (compare lanes 3 and 4 and lanes 6 and 7), in contrast to the results obtained in experiments with promoter DNA (Fig. 3), wild-type Eσ⁵⁴ was able to interact with wild-type PspF-(1–275) and PspF(T86S)-(1–275) as well as Eσ⁵⁴(G4L). Because σ⁵⁴ Region I adopts different conformations in the presence (i.e. within promoter complexes) and absence of promoter DNA (20, 21), it seems that the conformation adopted by σ⁵⁴ Region I in the presence of certain promoter DNA conformations is unfavorable for interaction with PspF(T86S)-(1–275). Clearly, the G4L substitution allows PspF(T86S)-(1–275) to overcome this defect. However, on heteroduplex promoter DNA (-10/-1 probe), it seems that the conformation adopted by σ⁵⁴(G4L) Region I is unable to recover the defect displayed by PspF(T86S)-(1–275) in transcription (Fig. 4). Significantly, the results imply overall that the conserved threonine residue in the GAFTGA motif of AAA activators has a role in sensing the conformation of σ⁵⁴ Region I within promoter complexes as directed by the DNA conformation.

DNA Sequences Downstream of the -12 Consensus Promoter Region Prevent PspF(T86S)-(1–275) from Interacting with the Closed Complex—Previously, we showed that, during open complex formation, AAA activators can be cross-linked to DNA sequences downstream (termed here to-be-melted DNA) of the -12 consensus promoter sequence (22). Our results implied that AAA activators potentially interact with the to-be-melted DNA at some step during open complex formation (22). In light of the present observations suggesting that PspF(T86S)-(1–275) is defective in efficiently and productively interacting with the Eσ⁵⁴ closed complex, but not with Eσ⁵⁴ per se (i.e. in the absence of DNA) (Fig. 5), we considered whether the to-be-melted DNA sequences downstream of the -12 consensus region might somehow interfere with PspF(T86S)-(1–275) and prevent it from interacting with the Eσ⁵⁴ closed complex. We therefore constructed promoter probes (probes A–F) in which we systematically moved the downstream DNA end points (Fig. 6A). The end points were chosen based on DNA positions to which PspF-(1–275) can be cross-linked within ADP·AlF_x-dependent ternary complexes (22).⁶ Native PAGE analysis revealed that Eσ⁵⁴ bound probes A–F equally well (Fig. 6B). Similarly, Eσ⁵⁴(G4L) bound probes A–D as well as wild-type Eσ⁵⁴. However, Eσ⁵⁴(G4L) revealed ∼2-fold higher affinity for probes E and F compared with wild-type Eσ⁵⁴ (Fig. 6B). Next, we measured the ability of Eσ⁵⁴ and Eσ⁵⁴(G4L) closed complexes formed on probes A–F to interact with wild-type PspF-(1–275) in an ADP·AlF_x-dependent manner and to form ternary complexes. As shown in Fig. 6C, no detectable differences were seen in the ability of wild-type PspF-(1–275) to interact with Eσ⁵⁴ and Eσ⁵⁴(G4L) closed complexes formed on probes A–F and to form ternary complexes. As expected (Fig. 3), PspF(T86S)-(1–275) was defective in efficiently forming the ternary complex on probes A and B in reactions containing wild-type Eσ⁵⁴ (Fig. 6C, lanes 13 and 14). On probes C–E, 30–35% of the DNA was in the ternary complex in reactions containing wild-type PspF-(1–275) (Fig. 6C, lanes 3–5). In contrast, only ∼13% of the DNA was in the ternary complex in reactions containing PspF(T86S)-(1–275) (Fig. 6C, lanes 15–17). Reactions containing Eσ⁵⁴(G4L) confirmed that the G4L mutation in Region I of σ⁵⁴ is able to rescue the ability of PspF(T86S)-(1–275) to form the ternary complex on probes A–E (Fig. 6C, lanes 19–23). Strikingly, however, PspF(T86S)-(1–275) was able to form the ternary complex on probe F as efficiently as wild-type PspF-(1–275) (Fig. 6C, compare lanes 15, 6, and 18). This implies that DNA sequence downstream of position -10 has a negative effect upon interactions between Eσ⁵⁴ and PspF(T86S)-(1–275). Interestingly, we also noted that ternary complex formation by both wild-type PspF-(1–275) and PspF(T86S)-(1–275) was relatively weaker on probe E than on all other probes in reactions with Eσ⁵⁴(G4L).

We extended the assay to investigate whether it is the non-template or template strand DNA that prevents PspF(T86S)-(1–275) from interacting with the closed complex. To do so, we constructed promoter DNA probes in which we shortened either only the non-template or template strand DNA. In each case, the end points were as described above (Fig. 6A). The results revealed that PspF(T86S)-(1–275) was unable to form the ternary complex if either the non-template or template strand DNA extended beyond position -10 (data not shown). Thus, it seems that the to-be-melted DNA sequences downstream of position -10, in either the single-stranded (template and non-template strands) or double-stranded conformation, somehow interfere with the ability of PspF-(1–275) to interact with the closed complex when the integrity of L1 is compromised by the T86S mutation.

DISCUSSION

Transcriptionally proficient open complex formation on bacterial promoters involves a complex and coordinated set of protein-protein and protein-DNA isomerization events. On σ⁵⁴-dependent promoters, such events are triggered in response to interaction of the closed complex with an AAA activator protein in an ATP hydrolysis-dependent manner. The highly conserved regulatory Region I domain of σ⁵⁴ is responsible for preventing open complex formation in the absence of activation. Accordingly, in the closed complex, Region I is positioned in the -12 consensus promoter region, where it makes repressive interactions with a fork junction DNA structure and so prevents open complex formation in the absence of activation (4). This nucleoprotein organization in the -12 promoter region is referred to as the “regulatory center” and constitutes a major interaction target for the AAA activator (3). Activation results in the reorganization of the regulatory center and the loss of interaction between σ⁵⁴ Region I and a repressive fork junction structure, and open complex formation then follows (1). The reorganization of the regulatory center is likely to occur via several discrete intermediate states. In a previous study, we proposed that the conserved GAFTGA motif of the AAA activator is part of an internal conformational signaling pathway and is involved in sensing and communicating the conformational variations adopted by the regulatory center en route to open complex formation (11). The substitution of the invariant threonine residue with serine in the GAFTGA motif prevents the AAA activator from sensing the conformation adopted by the regulatory center and results in a mutant AAA activator form that is incapable of efficient transcription activation both in vivo and in vitro. The key result of this study is the finding that a Gly-to-Leu substitution in Region I of σ⁵⁴ (position 4 in K. pneumoniae σ⁵⁴) is able to specifically recover the activation-defective property conferred by the Thr-to-Ser substitution in the highly conserved GAFTGA sequence of the AAA activator PspF (and NifA). Thus, the results from the set of experiments presented here clearly point toward an involvement of σ⁵⁴ Region I in the sensing and communicating of information relating to regulatory center conformation by the GAFTGA motif during open complex formation. The interplay between σ⁵⁴ Region I and the GAFTGA motif during open complex formation is of functional significance because σ⁵⁴ Region I also appears to determine the activities of three structurally conserved RNAP domains (β′ lobe, β′ jaw, and β′ clamp) that contribute to a DNA-binding channel in RNAP, where DNA downstream of the RNAP active center lies, and ensure that stable DNA opening near the transcription start site is maintained (7, 23). Previously, we proposed that σ⁵⁴ Region I acts as a “relay” domain and communicates with the downstream DNA-binding channel in RNAP in response to activation (24). In view of the present results, we suggest that σ⁵⁴ Region I could also be involved in relaying information regarding the regulatory center conformation to the AAA activator during transcription activation.

On pre-melted DNA, which mimics the conformation of the promoter in the open complex, σ⁵⁴(G4L) is unable to recover the activation defect of PspF(T86S)-(1–275) (Fig. 4). However, unlike wild-type σ⁵⁴, σ⁵⁴(G4L) allows PspF(T86S)-(1–275) to interact with the Eσ⁵⁴·pre-melted DNA complex. Because the pre-melted DNA does not support transcription in the absence of activation, the regulatory center of this DNA most likely adopts the repressed (non-activated) conformation (25). In contrast, the pre-melted DNA is in the “open complex” conformation. Thus, the fact that σ⁵⁴(G4L) allows PspF(T86S)-(1–275) to interact with but not activate Eσ⁵⁴ for abortive initiation on the pre-melted DNA further argues for a role for the GAFTGA motif in sensing and communicating DNA conformation. Notably, the results with the pre-melted DNA also imply that the leucine substitution does not simply contribute to a non-native interaction with the GAFSGA motif of PspF(T86S)-(1–275), which facilitates its interaction with the regulatory center. Rather, it seems that σ⁵⁴(G4L) with the appropriate promoter DNA structure can utilize PspF(T86S)-(1–275).

PspF(T86S)-(1–275) is unable to interact stably with the closed complex, but is able to interact with Eσ⁵⁴ in the absence of promoter DNA. Experiments with shortened promoter probes (Fig. 6) indicated that the DNA downstream of position -10, i.e. the to-be-melted DNA that lies downstream of the regulatory center, prevents the AAA activator from interacting with the closed complex if the integrity of the GAFTGA motif is compromised by the Thr-to-Ser substitution. In the closed complex, σ⁵⁴ interacts with the DNA to position -5 (26). Thus, on shortened probes, e.g. probes E and F (Fig. 6A), the lack of or an altered interaction between σ⁵⁴ and the DNA to position -5 could permit PspF(T86S)-(1–275) to interact with the regulatory center. The DNA downstream of position -10 is nonconserved in σ⁵⁴-dependent promoters, but appears to influence the functionality of the AAA activator via the GAFTGA motif. Thus, a direct interaction between PspF-(1–275) and the to-be-melted promoter DNA is possible. A previous study has reported that the AAA activator lies proximal to the to-be-melted DNA during transcription activation (22). However, the precise role of the to-be-melted DNA and the nature of the proximity of the AAA activator to this DNA segment in transcription activation remain unclear. Transcriptional silencing of the closed complex in the absence of activation strictly relies upon the interaction made predominantly by σ⁵⁴ Region I with a fork junction DNA structure at position -12/-11 within the regulatory center, which masks the determinants in σ⁵⁴ needed for binding downstream single-stranded DNA between positions -10 and -7 during early stages of open complex formation (27). ATP hydrolysis-dependent activation of the closed complex by the AAA activator appears to unmask the determinants in σ⁵⁴ needed for single-stranded DNA binding and to allow σ⁵⁴ to establish interactions with single-stranded DNA for open complex formation (27). We propose that, during activation, the AAA activator could nonspecifically sense the conformation of the DNA downstream of position -10 and couple the DNA structure per se to σ⁵⁴ Region I (and possibly to other determinants of σ⁵⁴ and RNAP) or couple information relating to its conformation to σ⁵⁴ via Region I to allow single-stranded DNA-binding determinants in Eσ⁵⁴ to establish the interaction with the single-stranded DNA segments needed for open complex formation. The transcription activation defect in AAA activators with a Thr-to-Ser substitution in the GAFTGA motif could manifest itself through their defective and negative interactions with the DNA downstream of position -10. The results here suggest that the absence of DNA downstream of position -10 permits PspF(T86S)-(1–275) to directly interact with the regulatory center. The characterization of σ⁵⁴(G4L) did not reveal any obvious (activation-independent) gain of single-stranded DNA-binding properties (in the context of the holoenzyme) compared with wild-type σ⁵⁴. Thus, we propose that the G4L mutation most likely somehow alters the conformation of the regulatory center and thereby enables PspF(T86S)-(1–275) to interact and communicate with the regulatory center independently of DNA downstream of position -10 or with a binding energy that overcomes the negative effects of DNA downstream of position -10.

The functional state of PspF-(1–275) (and most AAA activators) is a hexamer (10). How many GAFTGA motifs are involved in interactions with the regulatory center during transcription activation remains unclear. Cryoelectron microscopic analysis of the PspF-(1–275)·σ⁵⁴ complex suggested that at least two GAFTGA motifs interact with σ⁵⁴. The results here now suggest that the GAFTGA motif of the AAA activator has multiple interrelated roles during transcription activation. In the crystal structure of PspF-(1–275) (9, 10) and A. aeolicus NtrC1 (12), the GAFTGA motif is located on the tip of a flexible loop (L1) that juts out during ATP hydrolysis and contacts the regulatory center. Thus, it is possible that more than one GAFTGA motif interacts with the regulatory center (thereby also sensing/communicating, via/to σ⁵⁴ Region I, the different conformation adopted by the regulatory center) during successive rounds of ATP hydrolysis to allow the regulatory center to proceed through the range of conformational changes required for progression to an open complex formation. Overall, the results presented here highlight the level of regulatory precision and complexity that operates during transcription activation on σ⁵⁴-dependent promoters.