Introduction

Top Abstract Introduction Results Discussion Materials and methods References

Transcription by RNA polymerase (RNAP) is often regulated by interactions with control proteins to link specific gene expression to environmental signals and temporal cues. Often activators help recruit RNAP to promoters to increase initiation rates (Busby and Ebright 1999). In contrast, activity of the bacterial ⁵⁴ containing RNAP holoenzyme is regulated at the DNA melting step (for review, see Buck et al. 2000). Hydrolysis of an NTP by an activator drives a change in configuration of the ⁵⁴-holoenzyme, converting the initial closed complex to an open complex to allow interaction with the template DNA for mRNA synthesis (Wedel and Kustu 1995). Preopening of DNA templates does not overcome the requirement for NTP hydrolysis by an activator to promote engagement of the holoenzyme with the melted DNA (Wedel and Kustu 1995; Cannon et al. 1999).

The activators of ⁵⁴-holoenzyme are members of the large AAA+ protein family, which use ATP binding and hydrolysis to remodel their substrates (Neuwald et al. 1999; Cannon et al. 2000, 2001). The greater part of the central domain of ⁵⁴ activators corresponds to the AAA core structure, and includes ATP-binding and hydrolyzing determinants. The ⁵⁴ protein is known to be the primary target for the NTPase of activators, but how activators use NTP binding and hydrolysis is not well understood (Cannon et al. 2000). Similarly, the nature of the interaction between ⁵⁴ and the activator is not well described, but an interaction with ⁵⁴ can be detected in the case of the DctD activator by protein cross-linking (Lee and Hoover 1995). Here we show that the use of ADP-aluminum fluoride, an analog of ATP that mimics ATP in the transition state for hydrolysis, allows formation of a stable complex among the activator PspF, the PspF and NifA central activating domains, and ⁵⁴. The binding assay was used to help define determinants in ⁵⁴ and the activator needed for their interaction, and to show that binding can lead to an altered ⁵⁴-DNA footprint. The need for a transition-state analog of ATP for protein-protein binding is discussed in relation to the required ATPase activity of activators of ⁵⁴-dependent transcription. In particular, it seems that altered functional states of activators exist as ATP is hydrolyzed. This suggests a parallel to some switch and motor proteins that use nucleotide binding and hydrolysis to establish alternate functional states (Hirose and Amos 1999).

	Results

Top Abstract Introduction Results Discussion Materials and methods References

Assay system

Enhancer-binding activators of the ⁵⁴-holoenzyme are typically composed of three domains (Drummond et al. 1986; Morett and Segovia 1993). These include a C-terminal enhancer DNA-binding domain and an N-terminal domain. The latter functions in regulation, often by acting on the central domain (Lee et al. 2000). Interactions with the ⁵⁴-holoenzyme and ATP-binding and hydrolyzing activities directly involve the activator central domain. The PspFHTH protein we have employed here represents mainly the central domain of the ⁵⁴ activators (Fig. 1a). The PspF activator of Escherichia coli lacks a regulatory N-terminal domain, being subject instead to control by PspA (Jovanovic et al. 1999; Dworkin et al. 2000). Interactions of ⁵⁴ and the activator PspFHTH were explored in the presence of MgADP and compounds that are known to mimic the transfer of the -phosphate at hydrolysis of ATP (transition state analogs) with several proteins, as defined by X-ray crystallography of the nucleotide-containing complexes (Fersht 1998). In particular, we were interested in exploring the possibility of isolating ⁵⁴-activator complexes that depend on nucleotide interactions with the activator. The basic assay consisted of incubating the activator with the transition state analog ADP-aluminum fluoride (ADP · AlF_x) together with ⁵⁴ (or holoenzyme), or a DNA complex thereof, and resolving the mixture on a native polyacrylamide gel. Typically either one of the protein components or DNA was ³²P-end labeled, and in some experiments complexes were visualized by Coomassie staining.

View larger version (45K): [in this window] [in a new window]   Figure 1.   (a) Schematics of 54 and the activator PspF. The three regions of Klebsiella pneumoniae 54 and their associated functions are indicated (Buck et al. 2000). The functional domains of the Escherichia coli 54 activator PspF and its derivative PspFHTH deleted for its DNA-binding domain are shown (Jovanovic et al. 1999). The approximate position of the highly conserved GAFTGA motif implicated as part of the Switch 1 region in 54 activators (Rombel et al. 1998; Yan and Kustu 1999) is indicated. (b) Gel mobility-shift assay for ADP-aluminum fluoride-dependent complex formation between PspFHTH and 54 using 32P-HMK-tagged protein. Reactions were with 32P-HMK-tagged 54 or 32P-HMK-tagged PspFHTH (100 nM), unlabeled PspFHTH (10 µM), and unlabeled 54 (1 µM). Lane 10 contains 54 (1 µM), PspFHTH (10 µM) with 32P-end-labeled Sinorhizobium meliloti nifH promoter 88-nt DNA (16 nM). Arrow (a) indicates position of complexes formed between 54 and PspFHTH in the presence of ADP · AlFx; arrow (b) indicates the position of PspFHTH complex formed in the presence of ADP · AlFx, and arrow (c) indicates the position of DNA-54-PspFHTH complex in the presence of ADP · AlFx. (c) Gel mobility-shift assay for ADP · AlFx-dependent complex formation between PspFHTH and 54 and 54-holoenzyme detected by Coomassie staining. Reaction conditions were as in a except for PspFHTH (20 µM), 54 (4 µM or 600 nM when present with core RNAP [E]), and core RNAP (300nM). The arrow on the gel indicates the holoenzyme trapped activator complex in lane 6. Arrows (a) and (b) point to complexes as indicated in a. (d) Wild-type PspF and the NifA central domain form an ADP · AlFx-dependent complex with 54. Reaction conditions were as in b with 32P-HMK 54 (50 nM), PspFHTH (10 µM), wild-type PspF (3 µM), and NifA central domain (3 µM). PspFHTH (~36 kD), wild-type PspF (~37.5 kD), and NifA-CD (~32 kD).

ADP-aluminum fluoride induces formation of a stable complex between the activator and ⁵⁴

Initially either ⁵⁴ or PspFHTH was ³²P-end-labeled through an engineered heart muscle kinase (HMK) tag (Casaz and Buck 1997). Under conditions where ADP · AlF_x (where x must be 3 or 4) can form, but not otherwise, the end-labeled protein (either ⁵⁴ or PspFHTH) was found in a new, slow-running complex when the nonlabeled protein (activator or ⁵⁴, respectively) was added (Fig. 1b, lanes 3,8). Similar results were also achieved using proteins lacking the heart muscle kinase tag and in the absence of -lactoalbumin (a nonspecific carrier protein, see Materials and Methods), with complexes being detected by Coomassie staining (Fig. 1c). PspFHTH in the presence of ADP · AlF_x also bound ⁵⁴-holoenzyme (E⁵⁴; Fig. 1c, lane 6). Results show that ⁵⁴ and its holoenzyme can detectably associate with PspFHTH to form a stable complex in the presence of ADP · AlF_x. Hereafter we use the term "trapped" to refer to the form of activator bound to ⁵⁴ or ⁵⁴-holoenzyme in the presence of ADP · AlF_x.

Controls using core RNAP (E) alone did not result in the increased formation of a complex between core RNAP and PspFHTH-ADP · AlF_x, suggesting that ⁵⁴ is the main target of the activator within the holoenzyme (Fig. 1c, cf. lanes 3 and 8 with lane 4). Interestingly, PspFHTH can interact with core RNAP in the absence of ADP · AlF_x (Fig. 1c, cf. lane 3 with lanes 1, 7, and 11).

We also used ADP · AlF_x with the full-length PspF activator (i.e., with its DNA-binding domain) and the central domain of the nitrogen fixation A protein, NifA-CD (Money et al. 2001), another activator of the ⁵⁴-holoenzyme, so as to trap stable complexes with ⁵⁴ (Fig. 1d). As predicted from the presence of the activator PspFHTH within the trapped complex that formed with ⁵⁴, and the different molecular weights of the PspFHTH, PspF, and NifA-CD, these three ⁵⁴ trapped complexes each had a different native gel mobility (Fig. 1d).

Order of addition experiments in which either ³²P-⁵⁴ (50 nM) and PspFHTH (10 µM) were preincubated prior to formation of ADP · AlF_x (as in the standard reaction; see Materials and Methods) or PspFHTH was exposed to ADP · AlF_x before addition of ⁵⁴, resulted in 24% and 1% of the ³²P-⁵⁴ bound in the trapped complex, respectively. Formation of the ⁵⁴-holoenzyme trapped complex was subject to the same order of addition effects (data not shown). This strongly suggests that the transition-state analog ADP · AlF_x acts to stabilize a preexisting unstable complex between ⁵⁴ and PspFHTH.

Addition of 20 mM phosphate or 10 mM ATP after trapped complexes had been allowed to form did not diminish the amount of trapped ⁵⁴-PspFHTH complex, indicating that the ADP · AlF_x is stably bound in the complex (data not shown). ADP without aluminum fluoride, use of the alternative transition-state analog ADP · Vi (ADP in the presence of vanadate ion), or nonhydrolyzable analogs AMPPNP or ATPS did not result in formation of a stable ⁵⁴-PspFHTH complex (data not shown; Cannon et al. 2000, 2001). Other sigma factors (E. coli ⁷⁰ or ³⁸) did not associate with PspFHTH-ADP · AlF_x to give the slow-migrating trapped complex (data not shown). Therefore, We conclude that ADP · AlF_x acts specifically to increase the binding of activator to ⁵⁴ and its holoenzyme.

Using Coomassie staining we estimated the amount of ⁵⁴ and PspFHTH in trapped complexes isolated from a native gel (data not shown). Repeated experiments indicated that not less than five PspFHTH monomers are present per ⁵⁴ monomer. This implies that an oligomeric form of activator binds to ⁵⁴. Simple steric effects may also limit the number of ⁵⁴ molecules bound per activator oligomer.

ADP · AlF_x changes self-association and ATPase activity of PspFHTH

The native gel mobility of the PspFHTH activator is changed when ADP · AlF_x is allowed to form (Fig. 1b, cf. lanes 7 and 9; Fig. 1c, cf. lanes 7 and 8). This could be caused by differences in oligomerization state and/or conformation. Activators, in particular NtrC, of the ⁵⁴-holoenzyme are known to form higher-order oligomers (Wyman et al. 1997), and this is also true for PspF and PspFHTH (see below). Activators of ⁵⁴ belong to the AAA+ protein family (Neuwald et al. 1999; Vale 2000), crystal structures of which show nucleotide interactions in one protomer and contact to the -phosphate from an adjacent protomer within a hexameric assembly (Neuwald et al. 1999). Preliminary gel filtration experiments and analytical ultracentrifigation analyses have shown that ADP · AlF_x does increase the association state of the PspFHTH protein (data not shown). Because the PspF protein is known to interact with ATP (Jovanovic et al. 1999), we infer that the self-associated activator is in an ADP · AlF_x-bound form. The ATPase activity of PspFHTH and PspF were inhibited by ADP · AlF_x. With PspFHTH (3.0 µM) or PspF (1.0 µM) and ATP (0.4 mM), the presence of ADP · AlF_x reduced ATPase activities by 40% and 95%, respectively. ⁵⁴ is not known to interact directly with nucleotides, suggesting that binding of ⁵⁴ to PspFHTH is stabilized through interactions made between ADP · AlF_x and activator.

Role of activator self-association in binding ⁵⁴

Trapping experiments were performed using wild-type PspF at concentrations above that at which it fully self-associates and forms a higher-order oligomer (data not shown). Addition of ADP · AlF_x did not alter the native gel mobility of the wild-type PspF but did allow it to bind ⁵⁴ (data not shown; Fig. 1d, lane 4). Addition of ATP, ADP, or ATPS did not allow wild-type PspF to bind stably to ⁵⁴ (data not shown). Formation of a higher-order oligomer per se does not therefore allow PspF to stably bind ⁵⁴. Rather, a distinct form of PspF associated with the presence of the transition-state analog ADP · AlF_x is required for a stable interaction between activator and ⁵⁴. The ADP · AlF_x-dependent self-association of PspFHTH may reflect loss of a contribution by the HTH to self-association that allows the effects of binding the ATP analog to be visualized in terms of oligomerization changes. Binding of ADP · AlF_x between protomers can help account for the self-association of PspFHTH.

⁵⁴ Region I is essential for binding activator

⁵⁴ fragments 57-477 (⁵⁴ deleted for Region I, I⁵⁴), 1-324, 70-324, and a series of ⁵⁴ Region I three alanine substitution mutants from residue 6 to 50 (Casaz et al. 1999), both alone or as part of the holoenzyme, were screened for trapped complex formation with end-labeled PspFHTH activator (Fig. 2a; data not shown). It is clear that the ⁵⁴ N-terminal Region I sequences (residues 1-56) are important for the binding reaction with PspFHTH-ADP · AlF_x. No single three alanine substitution mutant in ⁵⁴ Region I diminished formation of the trapped complex with ⁵⁴ or ⁵⁴-holoenzyme as greatly as did removal of Region I (Fig. 2a; data not shown). However, clear patterns of reduced binding were apparent, suggesting that several sequences in Region I contribute to binding of the activator (Casaz et al. 1999). With ³²P-HMK-tagged PspFHTH at 100 nM and ⁵⁴-holoenzyme at 300 nM, Region I residues 6-11, 33-38, and 45-47 stood out as important patches for binding PspFHTH-ADP · AlF_x to holoenzyme. Triple alanine substitutions across these positions bound 20%, 30%, and 50% of the PspFHTH-ADP · AlF_x compared to wild-type holoenzyme, respectively (data not shown). For residues 33-38 and 45-47, binding activity correlates with the critical role of these patches in activated transcription (Syed and Gralla 1998; Casaz et al. 1999; Gallegos and Buck 2000). Importantly, two mutants in ⁵⁴ (deletion 310-328 and R336A) that share with certain ⁵⁴ Region I mutants and the Region I deletion form of ⁵⁴ the property of activator-independent transcription in vitro (Chaney and Buck 1999; Chaney et al. 2000), efficiently formed trapped complexes with PspFHTH (data not shown). This further supports the argument that ⁵⁴ Region I may directly interact with PspFHTH. Experiments using ⁵⁴ fragments 70-324 and 1-324 together with I⁵⁴ (residues 57-477) and wild-type ⁵⁴ showed that the NifA-CD had the same specificity for Region I as did PspFHTH in the trapping reaction (Fig. 2a; data not shown).

View larger version (43K): [in this window] [in a new window]   Figure 2.   (a) Gel mobility-shift assay for ADP · AlFx-dependent complex formation between PspFHTH and 54, 54 peptides, and 54-holoenzyme with and without Region I. Reactions contained 32P-HMK-tagged PspFHTH (100 nM), 54, and I54 (1 µM). Peptides 1-324 and 70-324 (50 µM). E54 and EI54 were formed with 54 (600 nM) and E (300 nM). Trapped activator-54 fragment complexes are marked with an arrow. (b) Gel mobility shift assay for ADP · AlFx-dependent complex formation between PspFHTH and 54 Region I. Reactions contained 32P-HMK-tagged PspFHTH (100 nM) and 54 Region I (50 µM). The lower, unfilled arrowhead indicates the PspFHTH-ADP · AlFx complex and the upper, filled arrowhead the trapped PspFHTH-Region I complex. (c) V8 footprinting of the trapped PspFHTH-54 complex. Reactions contained 32P-HMK-tagged 54 (200nM) and PspFHTH (20 µM). V8-treated reactions are marked with + (lanes 2,4,6,6`) and untreated reactions are marked with  (lanes 1,3,5,5'). Lanes 5' and 6` contain the free 54 isolated from reactions in lanes 5 and 6, respectively. V8 cleavage sites are as marked.

Interactions of ⁵⁴ Region I with activator

To explore the possibility that Region I (residues 1-56 of ⁵⁴) sequences might directly bind activator, we added purified Region I to PspFHTH (Fig. 2b). A distinct, buffer-independent, small reduction in gel mobility was seen when PspFHTH-ADP · AlF_x was formed in the presence of Region I, compared to controls without Region I (Fig. 2b, cf. lanes 2 and 3). No stable interaction was seen between PspFHTH and Region I in the absence of ADP · AlF_x (Fig. 2b, lane 1; data not shown). This result provides direct evidence for a PspFHTH-ADP · AlF_x-Region I interaction. The absence of other regions of the ⁵⁴ protein may allow Region I to interact with the PspFHTH monomer so as to inhibit activator self-association in the presence of ADP · AlF_x. This could explain why lane 3 does not also contain a band with the mobility corresponding to self-associated activator as seen in lane 2. 

View larger version (56K): [in this window] [in a new window]   Figure 3.   Gel mobility-shift assay for ADP · AlFx-dependent complex formation between the PspFHTH T86S GAFTGA mutant and 54 detected by Coomassie staining. Reactions contained PspFHTH T86S (20 µM) and 54 (2 µM). Arrow (a), trapped 54-PspFHTH T86S complex; (b), PspFHTH T86S-ADP · AlFx complex.

Experiments using heterobifunctional cross-linking reagents revealed that determinants for nucleotide-independent binding of ⁵⁴ to the activator DctD were outside of ⁵⁴ Region I (Lee and Hoover 1995; Kelly et al. 2000). Therefore, we performed a competition assay wherein an increasing concentration of Region I or I⁵⁴ was added to a fixed amount of ⁵⁴ and PspFHTH. At a ratio of 4:1 (Region I or I⁵⁴:³²P-⁵⁴) added prior to trapping, Region I reduced the amount of ⁵⁴ in the trapped complex by 68% and I⁵⁴ reduced the amount by 20% (data not shown). Together these results suggest that Region I is the region of primary contact between PspFHTH and ⁵⁴ before and after trapping but that additional determinants for an activator-⁵⁴ interaction prior to trapping do exist outside of Region I. Consistent with the Region I-trapped activator interaction assays, protein footprints of the stable PspFHTH-⁵⁴ complex formed with ADP · AlF_x showed that much of the Region I sequence was protected from protease attack (Fig. 2c, lane 6). In contrast, unbound ⁵⁴ from the same trapping and footprinting reaction was not protected across Region I (Fig. 2c, cf. lanes 6 and 6`). Protection in trapped complexes extended as far as amino acid 135, within the acidic Region II of ⁵⁴. Overall we conclude that PspFHTH-ADP · AlF_x and ⁵⁴ form a complex that involves direct protein-protein contacts between Region I of ⁵⁴ and the activator.

⁵⁴ mutants implicate interactions between the GAFTGA motif and Region I in vivo

A signature of activators of the ⁵⁴-holoenzyme is the six-amino-acid GAFTGA motif within the C3 region, which is involved in transcriptional activation and implicated in energy coupling (Morett and Segovia 1993; Wang et al. 1997; Gonzalez et al. 1998; Rombel et al. 1998). In an attempt to identify the determinants of the ⁵⁴-holoenzyme involved in the interaction with activator proteins, we searched for mutants of ⁵⁴ able to recover activator function of activation-defective mutants in the GAFTGA motif and in an adjacent residue in the C3 region of Bradyrhizobium japonicum nifA (Gonzalez et al. 1998). This strategy is based on the premise that in a macromolecular assembly the activity of a mutation that affects one of the members can be suppressed through a compensatory mutation in an interacting member.

Randomly generated mutants across Regions I and II of ⁵⁴ were screened for suppression of the NifA E298D (outside of GAFTGA) and NifA T308S (within GAFTGA) mutants, which give a low (<1% compared to wild-type NifA) transcription activity in vivo (Gonzalez et al. 1998). From an initial pool of ~50,000 mutants, two rounds of screening resulted in selection of a mutant that consistently maintained the suppression phenotype. The nucleotide sequence of this clone revealed six nucleotide changes, resulting in four amino acid replacements and two silent changes (Q20L, CAG to CTG; H53N, CAC to AAC; D89D, GAT to GAC; D159G, GAA to GGA; R196R, CGT to CGC; and D231V, GAC to GTC). To determine which amino acid replacements were responsible for the phenotype, the four amino acid changes were segregated. The suppression phenotype was only maintained when Q20L and H53N mutations were present simultaneously (clone Q20L/H53N in Table 1a). The data presented in Table 1a suggest that mutant ⁵⁴ Q20L/H53N suppressed mutant NifA T308S in an allele-specific manner, because its activity was increased 23-fold but the activity of NifA E298D was increased only fivefold. Expression in combination with the wild-type NifA does not seem to be affected.

View this table: [in this window] [in a new window]   Table 1.   -galactosidase activity from K. pneumoniae nifH and S. meliloti nifH promoters in an rpoN-background

The suppression potential of ⁵⁴ Q20L/H53N was also examined with region C3 GAFTGA mutants in PspF. To do so, the corresponding amino acid substitutions T308S and T308V of B. japonicum NifA were made in PspFHTH by site-directed mutagenesis, resulting in PspFHTH T86S and PspFHTH T86V. A Sinorhizobium meliloti nifH-lacZ fusion was used to determine activity because this promoter can be activated in trans by PspFHTH. The data in Table 1b show that the changes generated in the GAFTGA motif of PspFHTH also strongly affected the activation function, as in the case of NifA, and that the ⁵⁴ Q20L/H53N suppressed the PspFHTH T86S mutant. As previously observed, in vivo expression of the S. meliloti nifH promoter in the absence of a plasmid-borne activator is greater than the Klebsiella pneumoniae nifH promoter. This is likely caused by cross-activation at this strong promoter by other ⁵⁴ activators present in the cell. Interestingly, expression from ⁵⁴ Q20L/H53N in the absence of plasmids carrying PspFHTH was about twice that seen with wild-type ⁵⁴, suggesting that cross-activation is more efficient with ⁵⁴ Q20L/H53N.

To identify other possible combinations of amino acids that could result in the same or a clearer suppression phenotype, codons 20 and 53 of ⁵⁴ were mutagenized to saturation using a pair of oligonucleotides with NNG/C at these positions. The resulting mutants were screened for suppression of the NifA T308S mutation. Ten colonies were detected that displayed a suppressor phenotype with the NifA T308S mutant. The plasmids of selected colonies were segregated and retransformed and their -galactosidase activity was determined (Table 1c).

Nine out of the 10 ⁵⁴ suppressor mutants of NifA T308S had leucine at position 20, as did the original suppressor ⁵⁴ Q20L/H53N; the remaining mutant had Q20V. This indicates a clear requirement for a hydrophobic amino acid, leucine, at this position to suppress the NifA T308S phenotype. Moreover, the combination leucine/asparagine was selected several times with different codons for leucine, which discounts duplication of siblings. A range of functional substitutions at position 53, in addition to asparagine, suppressed the low transcription activity phenotype of NifA T308S (Table 1c). Position 53 seems to be more accessible to substitutions that lead to suppression than position 20. It is noteworthy that Q20L plus H53F had activity double that of the parental mutant (Table 1c). Although the double mutant had considerable activity with NifA T308A, no mutants were isolated that could recover the transcription activity of NifA E298D (data not shown).

Overall, the in vivo data provide strong indirect evidence for a functional interaction between the GAFTGA motif and Region I of ⁵⁴. Q20 and H53 lie within ⁵⁴ sequences that are protected from protease attack by PspFHTH-ADP · AlF_x (Fig. 2c) and that directly bind to PspFHTH-ADP · AlF_x (Fig. 2b). Taken together, these data suggest that the Q20L and H53F suppression phenotype may be caused by an enhanced affinity for activator.

The PspF GAFTGA motif is a determinant of ⁵⁴ activator binding

Using PspFHTH GAFTGA mutants with T  S, T  A, or T  V mutations (amino acid 86), we examined whether the integrity of this motif was required for binding to the ⁵⁴ protein and its holoenzyme under trapping conditions (see below). In an in vitro transcription assay that allowed only one round of transcription from the S. meliloti nifH promoter (10 nM) with ⁵⁴-holoenzyme (100 nM) and PspFHTH (4.0 µM), the T86S mutant gave 52% whereas the T86A and T86V mutants each gave less than 1% of the activity of the wild-type PspFHTH. The higher than expected activity of the T86S mutant compared to the in vivo data (Table 1b) may be explained in part by the effect of transcription reinitiation kinetics that are not measured in the single-round transcription assay. ATPase assays showed that at 2.0 µM protein monomer, the T86S, T86A, and T86V mutants had ATPase activities equivalent to wild type (data not shown). These data discount a simple defect in nucleotide binding. Trapping assays suggest that sequences within the GAFTGA motif function in binding ⁵⁴ Region I. The T86A and T86V mutants failed to give the characteristic self-associated complex seen with PspFHTH-ADP · AlF_x in the absence of ⁵⁴ and did not detectably form ADP · AlF_x-dependent complexes with ⁵⁴ or the holoenzyme (data not shown). The T  S mutant was defective for forming the ADP · AlF_x-dependent self-associated complex seen with PspFHTH in the absence of ⁵⁴ (cf. Fig. 3, lane 3 with Fig. 1c, lane 8) but did form trapped complexes with ⁵⁴ and holoenzyme (cf. Fig. 3, lane 4 with Fig. 1c, lane 2; data not shown). Binding of ⁵⁴ to the T86S mutant may stabilize its oligomeric state in the presence of ADP · AlF_x. Results with the T86 mutants correlate to the known defects in the GAFTGA motif for transcription activation and ⁵⁴ isomerization (Gonzalez et al. 1998; Cannon et al. 2000), and suggest they are closely linked to changes in binding one functional state of the activator that is established upon interaction with ADP · AlF_x. Clearly, although binding of ⁵⁴ need not be directly or exclusively to the GAFTGA motif, it critically involves it.

⁵⁴-DNA interactions in trapped complexes

To determine the DNA-binding properties of the trapped activator-⁵⁴ complexes and compare these to ⁵⁴, we conducted band shift and footprint assays. We wished to learn what the functional consequences of binding trapped activator were with respect to the DNA interacting properties of ⁵⁴ that are central to maintaining the closed promoter complex and to establishing the open promoter complex.

The trapped activator-⁵⁴ complex binds promoter DNA  PspFHTH lacks a DNA-binding domain and its use simplifies band-shift assays that employ DNA probes. We showed that ⁵⁴ bound to DNA probes derived from the S. meliloti nifH promoter also formed trapped complexes with PspFHTH (Fig. 4a). The new activator and ADP · AlF_x-dependent slow-running DNA complex has a slightly lower mobility than the DNA-free trapped PspFHTH-⁵⁴ complex (Fig. 1b, cf. lane 10 with lanes 3 and 8). Controls showed that PspFHTH activator alone could not band shift the DNA irrespective of the presence of the trapping conditions (data not shown) and that the mobility of the ⁵⁴-DNA complexes remained unchanged under trapping conditions when activator protein was omitted (data not shown). When I⁵⁴ (⁵⁴ lacking Region I) was bound to DNA it did not form a new complex in the presence of PspFHTH-ADP · AlF_x, as was the case for trapping reactions without DNA (Fig. 2a). Trapped complexes containing promoter DNA were able to form when ⁵⁴ was bound to DNA before trapping and when the ⁵⁴ activator-trapped complex was allowed to form prior to addition of DNA. Compared to homoduplex DNA, a 12 to 11 heteroduplex (early melted DNA), representing DNA melted early in transcription initiation, is bound six- to eightfold more strongly by ⁵⁴ (Cannon et al. 2000). The trapped complex did not show a loss of preference for binding the early melted DNA compared to the homoduplex probe, indicating that trapping does not greatly change ⁵⁴-early melted DNA interactions (Fig. 4a, cf. lanes 3 and 6). Because Region I sequences of ⁵⁴ direct its tight binding to early melted DNA (Cannon et al. 1999, 2000; Gallegos and Buck 1999; Guo et al. 1999), this activity appears unaltered when ⁵⁴ is bound by PspFHTH-ADP · AlF_x, even though the activator is interacting with Region I. 

View larger version (60K): [in this window] [in a new window]   Figure 4.   Gel mobility shift assay for 54 bound to DNA in the presence of PspFHTH ADP · AlFx. (a) Reactions contained 32P-end-labeled Sinorhizobium meliloti nifH promoter 88-nt DNA (16 nM), 54 (1 µM), and PspFHTH (10 µM). The three arrows indicate the multiple bands obtained with the 10/1 (late melted) heteroduplex DNA. (b) Mobility of the isomerized 54-DNA supershifted complex (lane 1; Cannon et al. 2000) compared with the trapped PspFHTH-54-DNA complex (lane 2). ATP was used for isomerization. (c) DNA molecules used in this experiment. The consensus GG and GC of the 54 binding sites are indicated by the vertical bars. Mismatched regions that create the early and late melted DNA templates are indicated.

The trapped complex produces an extended DNA footprint  To explore further whether the trapped activator-⁵⁴ complex interacts differently with promoter DNA, we conducted DNA footprinting of the trapped ⁵⁴-promoter complexes. Using DNase I we found that the footprint of the trapped complex on homoduplex DNA was extended compared to ⁵⁴, and was clearly increased toward the start of transcription (Fig. 5, lane 4a). Untrapped ⁵⁴-DNA complexes from the same trapping and footprinting reaction did not show this footprint extension (Fig. 5, lane 4b). Unlike the ATP hydrolysis-dependent extended footprint of ⁵⁴, which requires the use of the early melted heteroduplex (Cannon et al. 2000, 2001), the extended trapped complex footprint was not DNA template-dependent. The trapped complex gave a similar extended DNase I footprint on both the early melted and homoduplex DNA (data not shown). Similar extended footprints on both DNA templates were also observed with trapped ⁵⁴-holoenzyme complexes (Fig. 5, lane 7a; data not shown). We conclude that the extended footprint occurs as a consequence of activator interacting with ⁵⁴ Region I at the point of ATP hydrolysis. The extended footprint could be caused by changed ⁵⁴-DNA interactions and/or protection by the presence of PspFHTH at the downstream side of the promoter.

View larger version (54K): [in this window] [in a new window]   Figure 5.   DNase I footprints of trapped complexes bound to promoter DNA. (a) Complexes were formed with Escherichia coli glnHp2 homoduplex 88-nt DNA (100 nM, bottom strand end-labeled), 54 (1 µM) or its holoenzyme (E54, 100 nM), and PspFHTH (20 µM) in the absence or presence of ADP · AlFx. Samples were treated with DNase I (1.75 × 103 units; Amersham Life Sciences) for 1 min and the reaction was stopped by the addition of EDTA (10 mM). Bound and unbound complexes were separated and excised from a native gel (b) and the DNA was eluted into H2O overnight at 37°C. Equal amounts of DNA were denatured and then electrophoresed through a 10% denaturing gel. Additions to each binding reaction are indicated above each lane. (Lane 1) Untreated DNA; (lane 2) DNA alone treated with DNase I. Lanes 4a and 7a show extended DNase I footprints (dashed lines) from the trapped 54-DNA and 54-holoenzyme-DNA complexes shown in b (marked with an arrow in lanes 4 and 7, respectively). Because of the fragment sizes migrating close to the gel front it was not possible to precisely define the downstream end of the extended footprint in lane 7a. (Lanes 4b,7b) Footprints of untrapped 54-DNA and 54-holoenzyme-DNA complexes shown in b (lanes 4 and 7, respectively). (b) Native gel showing DNase I-treated complexes described in a. Additions to each binding reaction are indicated above each lane.

Trapped activator does not induce efficient single-stranded DNA binding or open complex formation  We investigated whether trapped activator can induce single strand DNA binding on a preopened DNA template by examining trapped holoenzyme interactions with the late melted heteroduplex DNA. Holoenzyme formed with wild-type ⁵⁴ is able to form a heparin stable complex on late melted S. meliloti nifH promoter DNA if activator and a hydrolyzable nucleoside triphosphate are present (Wedel and Kustu 1995; Cannon et al. 1999). Trapping of activator did not allow the ⁵⁴-holoenzyme to form a heparin stable complex on the late melted DNA, even when initiating nucleotide was present (data not shown). This suggests that the interaction of trapped activator with the ⁵⁴-holoenzyme does not induce all the conformational changes in the holoenzyme that allow stable interactions with melted DNA. Consistent with this, the ⁵⁴-holoenzyme bound to promoter DNA with heteroduplex from 7 to 3, from 5 to 1, or from 3 to 1 did not acquire heparin stability in the presence of activator and ADP · AlF_x (data not shown). It seems that although trapping can alter ⁵⁴-DNA interactions (Fig. 4a), these changes are not sufficient to lead to properties seen in activated complexes of the ⁵⁴-holoenzyme.

	Discussion

Top Abstract Introduction Results Discussion Materials and methods References

Activator-RNAP contacts are known to be critical for regulated transcription initiation, and activators of the ⁵⁴-holoenzyme catalyze formation of open promoter complexes through hydrolysis of a nucleoside triphosphate. We have shown, using the central ATP-hydrolyzing domains of the activators PspF and NifA together with wild-type PspF, that in the presence of ADP-aluminum fluoride a stable complex between the activator and ⁵⁴ or its holoenzyme is formed. It seems that we may have isolated a new functional state of the activator. The combination of ADP and ions (Al⁺³ and F) is likely to form a planar complex that mimics the atomic arrangement of the ATP -phosphate in the transition state (Wittinghofer 1997). We propose that this transition-state analog of ATP (ADP · AlF_x) is interacting with activator to allow adoption of a functional state that has increased affinity for ⁵⁴, compared to the nucleotide-free form of the activator, or when nonhydrolyzable analogs such as GTPS and ATPS are used. ADP did not substitute for its aluminum fluoride form, suggesting a critical role for the ATP -phosphate in establishing the conformation of activator that binds ⁵⁴. We infer that one role of ATP hydrolysis in activation of the ⁵⁴-holoenzyme is to promote formation of a functional state of the activator that tightly binds ⁵⁴. Hydrolysis of ATP would indicate this functional state is not long lived, and that Pi and ADP release return the activator to an alternate state that has decreased binding for ⁵⁴ (Fig. 4b).

Characteristics of the ⁵⁴-activator binding interaction

Because activators exist in different functional states depending on the progress of nucleotide hydrolysis, different sets of binding interactions between the activator and ⁵⁴ seem likely, and may relate to different steps in the process of formation of the open complex. ⁵⁴ Region I sequences function before open-complex formation to maintain the closed complex, and after to stabilize the open complex, suggesting that a series of linked interactions between the activator and ⁵⁴ Region I may occur and need not all be direct (Cannon et al. 1999; Gallegos et al. 1999; Gallegos and Buck 2000; Guo et al. 2000). The trapped ⁵⁴ and ⁵⁴-holoenzyme-PspFHTH complexes were only able to form efficiently when the proteins were allowed to associate prior to formation of ADP · AlF_x. This suggests a pathway in which ⁵⁴ and activator associate weakly prior to interaction with nucleotide. We also showed that ⁵⁴ deleted for Region I (I⁵⁴) was able to compete weakly with full-length ⁵⁴ for PspFHTH prior to trapping, implying that at least some of the interactions between activator and ⁵⁴ prior to trapping lie outside of Region I. Additional evidence for a range of interactions is provided by results of cross-linking assays between ⁵⁴ and the activator DctD (Lee and Hoover 1995; Wang et al. 1997; Kelly et al. 2000). In these experiments the determinants for binding ⁵⁴ to DctD were outside of ⁵⁴ Region I, and in the N-terminal half of the activator C3 region.

In our experiments the addition of ADP · AlF_x results in a new stable complex between ⁵⁴ and activator that directly requires the regulatory Region I of ⁵⁴ for its formation. This suggests that ATP hydrolysis is used to promote a new binding interaction between ⁵⁴ Region I and activator. The dependence on ADP · AlF_x for detecting the complex indicates that the stable complex with ⁵⁴ is normally transient and is not seen when using ATP because of the short life of the transition state of ATP at hydrolysis. One inference is that coupling ⁵⁴ and activator interactions to ATP hydrolysis can lead to high rates of transcription through rapidly directing ⁵⁴ to a new functional state suitable for open-complex formation.

Activator-nucleotide interactions

The changed native gel mobility of PspFHTH resulting from incubation with ADP · AlF_x supports the idea that nucleotide binding can change the quaternary structure of this mutant activator. Tight binding of the transition state of ATP by PspFHTH may stabilize formation of an oligomer through one protomer binding the nucleotide base while an adjacent protomer contributes a contact to the -phosphate. Implicit in this view is the idea that the activator senses the -phosphate of the hydrolyzable nucleoside triphosphate, and that hydrolysis and changing interactions with the -phosphate allow the activator to interconvert between different functional states. The ATP-bound form of the activator NtrC is reported to interact with ⁵⁴ in a manner dependent on the presence of the -phosphate (Guo et al. 2000). These interconversions may also involve the amino acids located in regions of the ATP-binding fold, and may include residues equivalent to those that are known to comprise the Switch 1 and Switch 2 sequences of GTP-hydrolyzing signaling proteins (Gamblin and Smerdon 1998; Rombel et al. 1998). There appears to be a strong similarity between activators of the ⁵⁴-holoenzyme and motor proteins and signaling switch proteins that use nucleotide binding and hydrolysis to interconvert between functional states with different affinities for their targets. In the case of activators of the ⁵⁴-holoenzyme, activator-⁵⁴ interactions that depend on ATP hydrolysis lead to an increased DNA interaction by ⁵⁴ to enable open complex formation (Cannon et al. 2000, 2001).

A notable property of the mutants in the GAFTGA motif of PSPFHTH was their failure to efficiently self-associate in the presence of ADP · AlF_x (Fig. 3; data not shown). The normal levels of ATPase activity and its unchanged dependence on activator concentration for these mutants (J. Schumacher, unpubl.) support the argument that the self-association and interactions of the mutant proteins with ATP for hydrolytic cleavage are largely intact. Differences seen with ADP · AlF_x could be related to alterations in interactions with ADP or a subtle defect in self-association at some step after the hydrolytic cleavage of ATP. Although the PSPFHTH T86S mutant gave little self-associated product in the absence of ⁵⁴ (Fig. 3), the level of trapped ⁵⁴-PSPFHTH T86S complex was normal, suggesting that ⁵⁴ stabilizes an interaction with ADP · AlF_x that occurs through the GAFTGA motif.

The tight binding of trapped activator with ⁵⁴ and holoenzyme seen in the absence of promoter DNA raises the possibility that the enhancer-bound activator might recruit the holoenzyme to promoters that are weak binding sites for the holoenzyme. However, careful kinetic analysis to include consideration of the short lifetime of the transition state of ATP hydrolysis and the dissociation rate of the closed complex would be required to show such an effect was meaningful when ATP was being hydrolyzed. Nevertheless, it is clear that one particular binding interaction between ⁵⁴ and activator only occurs efficiently when the activator is in a particular "on" state that is transiently created when ATP is being turned over.

The role of ⁵⁴ Region I

The regulatory Region I of ⁵⁴ was clearly a determinant in the formation of the stable trapped complex with activator. This is consistent with the central role Region I has in activated transcription and in controlling the DNA-binding properties of ⁵⁴ and its holoenzyme that partly distinguish the closed and open promoter complexes (Wang et al. 1995; Casaz and Buck 1999; Guo et al. 1999; Gallegos and Buck 2000; Pitt et al. 2000). Binding assays with ⁵⁴ Region I alone strongly suggest that activator makes a direct contact to it. Analysis of 15 triple alanine substitution mutants spanning Region I (amino acids 6-50) showed none had a defect as great as deletion of the entire Region I, implying multiple determinants. Region I sequences localize in the core RNAP near the active site for RNA synthesis (Wigneshweraraj et al. 2000) and over the promoter region that is near the start of DNA melting. We have termed this protein-promoter DNA focus the regulatory center of the holoenzyme closed complex (Wigneshweraraj et al. 2001). Mutations in Region I lead to changes in the holoenzyme and ⁵⁴-DNA interactions that can lead to activator-independent transcription in vitro (Wang et al. 1995; Syed and Gralla 1998; Casaz et al. 1999). The trapped state of the activator may therefore make a contact to ⁵⁴ Region I within the regulatory center to start to change the conformation of the protein components of the closed complex.

DNA interactions of the trapped  complex

In the light of the distinctive changes in ⁵⁴ binding to late melted DNA, it appears that binding of ⁵⁴ by the trapped activator may begin to change the DNA-binding properties of ⁵⁴. Experiments using DNA heteroduplex from 10 to 1 showed multiple banding with the trapped ⁵⁴-PspFHTH complex. This suggests that an altered interaction with start site proximal single strand DNA is possible when ⁵⁴ Region I is stably engaged with trapped activator. When ATP or GTP is used, the activator drives ⁵⁴ to melt DNA from 9 to 6 (Cannon et al. 2001). We attempted to see whether the ⁵⁴ trapped complex had resulted in any DNA strand denaturation, using KMnO₄ or diethylpyrocarbonate as a probe for DNA base unstacking. Unfortunately, the sensitivity of the protein complex to both KMnO₄ and diethylpyrocarbonate precluded any meaningful interpretation of the DNA footprints.

The extended promoter DNase I footprint we see with ⁵⁴ and holoenzyme trapped activator complexes could be directly caused by any single component in the complex being proximal to the DNA downstream of the 12 promoter DNA. Because Region I of ⁵⁴, the binding site for activator, is located over the 12 promoter element (Wigneshweraraj et al. 2001), activator bound to Region I could be responsible, at least in part, for directly blocking DNase I access. Two observations are consistent with this idea. First, the isomerized complexes that form with the ⁵⁴, activator, and hydrolyzable NTP and give extended DNase I footprints require the use of early melted DNA (Cannon et al. 2000), whereas the extended footprint in trapped complexes was evident with homoduplex and early melted DNA. Therefore, the extended footprint seen with the trapped complex may not be owing to ⁵⁴ isomerization. Rather, the footprint may reflect an intermediate state of ⁵⁴, not the fully isomerized form, bound to activator. Second, the isomerized ⁵⁴-DNA complexes bind core RNAP poorly, and holoenzyme does not efficiently form the isomerized complex (Cannon et al. 2001). This contrasts with the clear extended footprint seen with the trapped holoenzyme-activator complex on homoduplex and early melted DNA, again supporting the argument that the extended footprint may not be caused by ⁵⁴ isomerization but, rather, to the presence of activator downstream of the 12 promoter DNA.

The transition state-dependent interaction between activator and ⁵⁴ occurs prior to open complex formation

The full ATP-hydrolysis cycle by activator results in a remodeling of the ⁵⁴-holoenzyme-DNA complex, with conformational changes being evident in