From the Department of Biological Sciences, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom
Received for publication, February 6, 2003
, and in revised form, March 13, 2003.
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ABSTRACT |
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INTRODUCTION |
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factors bind double-stranded promoter DNA and promoter DNA structures with single-stranded and double-stranded DNA juxtaposed (fork junctions) that mimic the conformational state of the promoter DNA as it exists within the transcription bubble. In the case of enhancer-dependent closed complexes, two base pairs at positions 12 and 11 (where DNA melting originates), which are adjacent to the transcription start site proximal promoter element (GC region; Table Ia), are transiently melted (6). Interactions between
54 and this transient fork junction structure are repressive, keeping the
54-RNAP silent for transcription by inhibiting its ability to melt DNA and isomerize to form open complexes (7, 8).
54-RNAP binds tightly to the template strand of this transient fork junction structure and as a consequence fails to make interactions with the non-template strand adjacent to the 12 fork junction. The latter interaction is a critical feature of open complex formation by enhancer-dependent and -independent RNAPs (8, 9).
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The amino-terminal region 1 of 54 makes a major contribution to binding the fork junction structure at 12 (7, 8, 10, 11, 12). This nucleoprotein organization constitutes the direct binding target for AAA activators and is known as the regulatory center (12, 13). The AAA activators of the
54-RNAP couple the energy derived from ATP hydrolysis to remodel the regulatory center, relieving the inhibitory interactions with the template strand of the fork junction structure at 12 and promoting new interactions with the adjacent non-template strand required for forming open complexes (5, 8, 11, 14).
Open complex formation by the 70-RNAP and
54-RNAP proceeds via several intermediate states involving large conformational changes in the RNAP (15). However, very little information exists on the mechanisms that trigger changes in RNAP and promoter DNA that lead to transcription. Analysis of the mechanisms of transcription initiation by the
54-RNAP will extend our understanding further on the functioning of the RNAP as a complex molecular machine. Recently, we demonstrated that it is possible to "trap"
54 or the
54-RNAP bound to promoter DNA with the E. coli AAA activator phage shock protein F (PspF) in the presence of ADP aluminum fluoride (ADP·A1Fx), an analogue of ATP at the point of hydrolysis (13). Using heteroduplex forms of the Sinorhizobium meliloti nifH promoter probes containing fork junction structures at 12 and, for experimental simplicity, only the AAA domain of PspF (PspF1275) (Table Ib), we now report that nucleotide-dependent action of AAA activators on
54 or
54-RNAP significantly changes the interactions made with fork junction DNA at 12. Altered activator-dependent promoter DNA binding activities of
54 and
54-RNAP were observed in the presence of poorly or non-hydrolyzed forms of ATP (adenosine 5'-(
-thio)triphosphate (ATP
S) and ADP·AlFx, respectively), suggesting that several nucleotide bound states of the AAA activator, each capable of remodeling
54-RNAP prior to completion of the ATP hydrolysis cycle, exist and that ATP hydrolysis per se is not needed for the partial remodeling of the regulatory center. Evidence that a mixed nucleotide-bound (ATP and ATP
S) state of the activator resulted in altered remodeling activity was also obtained. Overall the results provide clear evidence that discrete functionalities are associated with different nucleotide bound states of PspF, which orchestrate open complex formation by the
54-RNAP during the ATP hydrolysis cycle by remodeling
54-RNAP-fork junction interactions. Hence by inference we suggest that other AAA proteins will have several distinct nucleotide-dependent functional states.
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EXPERIMENTAL PROCEDURES |
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Native Gel Mobility Shift AssaysBinding reactions were conducted at 30 °C in STA buffer (25 mM Tris acetate, pH 8.0, 8 mM Mg-acetate, 100 mM KCl, 1 mM dithiothreitol and 3.5%(w/v) PEG-6000). Where indicated, 54,
I
54, or mutant
54 were present at 1 µM and the
54-RNAP holoenzyme at 100 nM (formed with 1:4 ratio of core RNAP to
54).
54 proteins or
54-RNAP were incubated for 5 min with 16 nM promoter probe and nucleotides as required: ADP·AlFx (formed in situ by the addition of 0.2 mM AlCl3 to a mixture containing 0.2 mM ADP and 5.0 mM NaF; Ref. 13), ATP (2 mM), ATP
S (2 mM, unless otherwise stated), or ATP
S/ATP mixture (0.2/2 mM, unless otherwise stated). After addition of 10 µM PspF1275 (unless otherwise stated) the reactions were incubated for a further 10 min prior to resolving on a 4.5% native polyacrylamide gel. Native gels were run in 25 mM Tris, 200 mM glycine buffer (pH 8.6). Complexes were detected by PhosphorImager analysis. The error range for the amount of DNA band shifted was ±5%, depending upon the
54 and ATP
S preparations used. For comparative purposes when using variants in PspF or DNA the same
54 and ATP
S preparations were used.
DNA Footprinting AssaysBinding reactions (10 µl) were conducted as described above but with 100 nM promoter DNA probe and in STA buffer without dithiothreitol. Footprinting reagents were added as described (11, 14), reactions were terminated, and bound and unbound DNAs were separated on native polyacrylamide gels. DNA was then excised, processed, and analyzed on a denaturing 10% polyacrylamide gel. For ortho-copper phenanthroline (OP-Cu) footprinting, 0.5 µl of a solution of 4 mM ortho-phenanthroline, 0.92 mM CuSO4, and 0.5 µl of 0.116 M mercaptoproprionic acid were added to the reactions and incubated for 2 min. The reactions were terminated by the addition of 1 µl of 28 mM 2,9-dimethyl-1,10-phenanthroline. For DNase I footprints, 1.75 x 103 units of enzyme (Amersham Biosciences) was added to the reactions for 1 min, followed by addition of 10 mM EDTA to stop cutting. For KMnO4 footprinting, 4 mM fresh KMnO4 was added for 30 s, followed by 50 mM -mercaptoethanol to quench DNA oxidation. Gel-isolated DNA was eluted into 0.1 mM EDTA (pH 8) (for DNase I footprinting) or H2O (for OP-Cu and KMnO4 footprinting) overnight at 37 °C. KMnO4-oxidized DNA was cleaved with 10% (v/v) piperidine at 90 °C for 20 min. Recoveries of isolated DNA were determined by dry Cerenkov counting and equal numbers of counts were loaded onto 10% sequencing gels. Dried gels were visualized and quantified using a PhosphorImager.
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RESULTS |
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As shown in Fig. 1a, 54 does not detectably bind to promoter probes with mutant or missing template strand sequences at position 12 (probes 29; Ref. 11). In contrast, strong binding of
54 and
54-RNAP was evident on probe 1, which contains wild-type template strand sequences within the heteroduplex segment (Table Ia and Fig. 1, a and b, respectively). Strikingly, in the presence of PspF1275-ADP·AlFx, the binding activity of
54 to probes 2, 7, and 8 was significantly increased, suggesting that the stable complex formation between
54 and PspF1275-ADP·AlFx (hereafter called the trapped
54) has altered the DNA-binding activity of
54 (Fig. 1a). Similarly, the promoter DNA-binding activity of the
54-RNAP was altered in the presence of PspF1275-ADP·AlFx. As shown in Fig. 1b, we found that the PspF1275-ADP·AlFx-bound form of
54-RNAP (hereafter called the trapped
54-RNAP) bound to probes 2, 4, 7, and 8 more efficiently than the untrapped
54-RNAP. Overall, the results clearly demonstrate an apparent activator-dependent change in the DNA-binding activity of
54 and
54-RNAP that critically involves PspF1275-ADP·AlFx. Furthermore, as shown in Fig. 1, a and b, the binding of
54 and
54-RNAP to the promoter probe with wild-type template sequences at positions 12 and 11 (probe 1) was unchanged in the presence of PspF1275-ADP·AlFx, suggesting that the activator does not interact to produce an overall general increase in DNA binding. Rather, a change in binding activity is evident only on certain heteroduplex promoter probes. Controls where ADP·AlFx or PspF1275 was omitted showed that the change in DNA-binding activity was specific to conditions that led to formation of the stable association of PspF1275-ADP·AlFx with
54 or
54-RNAP (data not shown). A hallmark of
54-RNAP open complexes is their stability in the presence of heparin. The trapped
54-RNAP complex bound to promoter probes 19 were no more heparin-stable than
54-RNAP promoter complexes, indicating that the complexes had not fully isomerized to acquire heparin stability, but rather might represent an intermediate state (data not shown).
DNA Sequences Around Position 1 Are Important for the Binding of Trapped 54 and
54-RNAP to Promoter Probe 2
DNA footprinting data on promoter probe 1 have established that compared with the binary 54-DNA complexes, ADP·AlFx-dependent trapped
54-DNA complexes have extended DNase I footprints toward the transcription start site, as do ATP hydrolysis-dependent isomerized binary
54-DNA complexes (11, 13). This indicates that the extended
54-DNA interactions occur in response to interactions with certain nucleotide-bound forms of the activator. We used the promoter probe with the mutant template strand sequence (GT at positions 12 and 11 instead of TG; probe 2), to which trapped
54 and
54-RNAP bound most efficiently (Fig. 1) to further examine the increased DNA binding functionality of trapped
54 and
54-RNAP. By using a series of shortened variants of promoter probe 2 (probes 2A2F; Table II), we explored whether the changed DNA-binding activity seen above (Fig. 1) relied upon the transcription start site proximal DNA sequences. As shown in Table II, increased binding of the trapped
54 and trapped
54-RNAP complexes to promoter probes that extend downstream of (and including) position 1 occurred (probes 2A2D). However, no increased binding activity of trapped
54 and trapped
54-RNAP to promoter probes lacking sequences downstream of the 1 position was observed (Table II). This suggests that sequences upstream of 1 are required for the increased binding activity of the trapped
54 and
54-RNAP complex to promoter probe 2. Furthermore, binding assays with promoter probes 2G and 2H demonstrated that the DNA between positions 3 and 1 had to be in duplex form.
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Binding of Trapped 54 and
54-RNAP to Promoter Probe 2 Leads to Altered Interactions with Fork Junction DNA at 12
OP-Cu is a minor groove-specific DNA cleaving reagent that has been widely used to study 54-RNAP interactions with the 12 promoter region (6, 19). In closed promoter complexes formed with the
54-RNAP on homoduplex promoter probe (Table Ia), the minor groove at the 12 position is distorted and thus is susceptible to cleavage by OP-Cu (6). OP-Cu-mediated DNA cleavage at this position is not evident in open promoter complexes suggesting that the distortion distinguishes closed and open promoter complexes (6). We used OP-Cu footprinting to probe whether binding of trapped
54 and trapped
54-RNAP to promoter probe 2 changes
54 interactions with the fork junction structure at 12. To do so, we formed promoter complexes with
54 and
54-RNAP on probe 2 in the absence and presence of PspF1275-ADP·AlFx, treated them with OP-Cu, and separated the probe 2 bound and unbound complexes by native-PAGE. Promoter complexes were excised from native gels, the DNA was isolated and analyzed by denaturing-PAGE.
As shown in Fig. 2, OP-Cu treatment of naked promoter probe 2 revealed a hypersensitive site around position 12 (lane 2), as expected because of the heteroduplex segment at this position (Table Ia). This hypersensitive site was also evident in the weak complexes formed between 54 or
54-RNAP and probe 2 in the absence of PspF1275-ADP·AlFx (Fig. 2, lanes 5 and 6). This is striking because the OP-Cu-mediated DNA cleavage at 12 is significantly reduced in complexes formed between
54 or
54-RNAP and probe 1 (which contains wild-type template strand sequences in the heteroduplex segment; Table Ia) because of the tight binding of
54 or
54-RNAP to this heteroduplex segment (19). Interestingly, analysis of the complexes formed between trapped
54 or trapped
54-RNAP and probe 2 showed that the DNA cleavage around position 12 is strongly reduced (Fig. 2, compare lanes 3 and 4 with 5 and 6). Thus, the footprinting data establishes that
54 and
54-RNAP interactions near the 12 promoter region are altered in the presence of PspF1275-ADP·AlFx.
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Interaction of ATPS Bound PspF1275 with
54 Results in a Novel Promoter Complex
To increase our understanding of how new functional states of PspF1275 are created by nucleotide interactions, we explored whether changes in DNA-binding activity of 54 and
54-RNAP in the presence of PspF1275-ADP·AlFx are seen with other nucleotides. The poorly or non-hydrolyzable forms of ATP, ATP
S, and adenosine 5'-(
,
-imido)triphosphate (AMP-PNP) do not support productive open complex formation by
54-RNAP (20), but could influence prior steps. We compared the functional states of PspF1275 bound to ADP·AlFx, ATP
S, or AMP-PNP in assays using promoter probes 1 and 2 (Fig. 3). Strikingly, the presence of the ATP
S-bound form of PspF1275 (PspF1275-ATP
S) resulted in the formation of a novel complex on promoter probe 2 only (Fig. 3, lane 13). Interestingly, the novel complex migrated considerably faster than the trapped
54-DNA complex and thus appeared to differ significantly from the latter (Fig. 3, compare lanes 12 and 13). The addition of AMP-PNP, ATP, or GTP did not substitute for ATP
S in formation of the ATP
S-dependent complex on promoter probe 2 (Fig. 3 and data not shown). The complex (hereafter called the ATP
S complex), like the trapped
54-DNA complex relied upon promoter sequences between positions 3 and 1 (Table II). Thus, these initial results demonstrate that a new
54-DNA complex can be obtained that is dependent upon a certain fork junction structure at position 12 and PspF1275-ATP
S.
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The ATPS-Complex Is a Novel Binary
54-DNA Complex
At high concentrations of 54 and promoter probe 2, we observed a weak
54-DNA complex that formed independently of PspF1275-ADP·AlFx or PspF1275-ATP
S. This binary
54-DNA complex migrated faster than the trapped
54-DNA complex and the ATP
S-dependent complex (data not shown). Using activators of different molecular weights (full-length PspF1325, PspF1292, PspF1275, and heart muscle kinase-tagged PspF1292), we obtained no evidence that the ATP
S-complex was a ternary complex containing the activator (data not shown). We were also unable to demonstrate the presence of 32P-PspF1292 in the ATP
S-complex in reactions where sensitivity was not limiting (data not shown). From these results we conclude that PspF1275-ATP
S enables
54 to bind promoter probe 2 and form a stable binary complex that does not depend on the stable association of PspF1275. Because the formation of the ATP
S-complex and the trapped
54-DNA complex relied upon promoter probe 2 and was independent of the order of addition of
54, DNA, or PspF1275 (data not shown), we suggest that the PspF1275-ATP
S interacts transiently with
54 to change the DNA binding functionality of
54. The ADP·AlFx and ATP
S forms of PspF1275 are therefore distinct in that only the ADP·AlFx-bound PspF1275 forms a stable complex with
54 (13), but both share the property of altering the DNA-binding activity of
54 on promoter probe 2.
Specificity of the ATPS-Complex
We investigated the requirements for ATPS-complex formation. Initial experiments in which increasing amounts of ATP, ADP, AMP, (NH4)2SO4 (to mimic the PO4 at the point of ATP hydrolysis), AMP-PNP, guanosine 5'-(
-thio)triphosphate (GTP
S), or ATP
S were present demonstrated that ATP
S-complex formation was specific to the
S forms of the trinucleotide (data not shown). As shown in Fig. 4a, the amount of ATP
S-complex formation increased with ATP
S, and saturated at 1 mM ATP
S (20% ATP
S-complex). Increased
54-DNA binding was also observed when ATP
S was substituted with GTP
S (data not shown), although ATP
S was the most effective in promoting binding of
54 to promoter probe 2 (
5-fold better than GTP
S). The amount of ATP
S-complex formation also titrated with PspF1275 and
54 (Fig. 4, b and c, respectively). Increasing amounts of DNA did not increase the fraction of DNA in the ATP
S-dependent complex, suggesting that PspF1275-ATP
S acts on free
54 and not on its promoter DNA-bound form (data not shown). Furthermore, ATP
S-complex formation was dependent upon the integrity of the GAFTGA motif of PspF1275 (residues 8388), a signature motif in the AAA domain of
54 activators, which is involved in direct binding interactions with
54 (11, 13, 17). No ATP
S-complexes were detected when PspF1275 harboring the T86A, T86S, F85A, or F85L mutations within the GAFTGA motif were used (data not shown). Because these PspF1275 mutants were not defective for nucleotide binding, we conclude that the failure to form the ATP
S-complex arises from a defective interaction with
54 and not defects in nucleotide binding per se. As expected, attempts to form the ATP
S-complex with PspF1275 harboring mutations within motifs involved in nucleotide interactions or in ATP hydrolysis (Walker A and B motifs and the putative "arginine finger" residue) failed (data not shown).
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Interaction of 54 with a Mixed Nucleotide-bound Form of PspF1275 Results in a Novel
54-DNA Complex
Activators of the 54-RNAP must form oligomeric structures to hydrolyze ATP (5, 21, 22). The state of the bound nucleotide within each protomer of the oligomer during active ATP hydrolysis is not known. However, we considered that differences could exist between protomers, and attempted to form the ATP
S-complex in the presence of increasing amounts of ATP
S plus either 4 mM ATP or AMP. As shown in Fig. 5a, we observed that the amount of ATP
S-dependent complex formation was significantly increased in the presence of ATP, especially at low concentrations of ATP
S (e.g. 0.2 mM). This increase was not seen with AMP (recall ATP alone does not cause increased binding of
54 to promoter probe 2). This suggests protomers of PspF1275 differentially loaded with nucleotide were functioning together to either produce a more active oligomer, or produce differentially loaded oligomers that work together to remodel the DNA-binding activity of
54. Strikingly, we noticed a slight but distinctive difference in migration between the ATP
S-complex and the complex formed in the presence of ATP
S and ATP (Fig. 5b). Thus, it appears that in the presence of ATP and ATP
S, PspF1275 adopts a functional state that is different from the ATP
S-bound form, the action of which on
54 leads to the formation of a novel
54-promoter DNA complex (hereafter called the ATP
S/ATP-complex). In all the experiments involving the use of the ATP
S/ATP mixture, we combined 2 mM ATP with 0.2 mM ATP
S to give the maximum amount of ATP
S/ATP-complex at low concentration of ATP
S (Fig. 5c).
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ATPS- and ATP
S/ATP-Complex Formation Requires the Same DNA and Protein Determinants
Assays with 32P-labeled PspF1292 confirmed that the ATPS/ATP-complex is a binary
54 complex (data not shown). Unsuccessful attempts to form the ATP
S/ATP-complex with PspF1275 mutants in the GAFGTA motif, in the Walker A motif or Walker B motif suggest that, like the ATP
S-complex, formation of the ATP
S/ATP-complex requires a form of PspF capable of hydrolyzing ATP and interacting with
54 (data not shown). Interestingly, the mixed nucleotide bound state of PspF1275 (hereafter called PspF1275-ATP
S/ATP) was still able to hydrolyze ATP as directly observed in ATPase assays (data not shown). AMP did not substitute for ATP in the ATP
S + ATP reactions (Fig. 5a). Furthermore, ADP was almost as efficient as ATP in working in combination with ATP
S to increase the number of
54-probe 2 complexes (data not shown). These results imply that the hydrolysis of ATP could be required for the ATP
S/ATP-complex formation. Overall, these results suggest that (i) the in situ formation of ADP + Pi and (ii) the presence of ATP
S in the nucleotide binding pockets of PspF1275 are important for the formation of the ATP
S/ATP-complex.
To investigate whether PspF1275-ATPS or PspF1275-ATP
S/ATP had any effect on the DNA-binding activity of
54-RNAP, we formed
54-RNAP promoter complexes in the absence and presence of PspF1275-ATP
S and PspF1275-ATP
S/ATP on probe 2 (Fig. 5d). Interestingly, as seen above with
54, the mobility of the
54-RNAP promoter probe 2 complex on native gels was changed in the presence of PspF1275-ATP
S and PspF1275-ATP
S/ATP (Fig. 5d, compare lane 2 with lanes 3 and 4). Control reactions with PspF1275 bound to AMP or AMP-PNP confirmed that the mobility change of
54-RNAP occurred in response to PspF1275-ATP
S or PspF1275-ATP
S/ATP (data not shown).
Action of PspF1275-ATPS and PspF1275-ATP
S/ATP on
54 and
54-RNAP Leads to Altered Interactions with the Fork Junction at 12
We first attempted to characterize the interactions between the fork junction promoter structure and 54 or
54-RNAP in the PspF1275-ATP
S and PspF1275-ATP/ATP
S complexes with probe 2 by OP-Cu and potassium permanganate (KMnO4) footprinting techniques (Fig. 6, a and b). KMnO4 is a single-stranded thymine-reactive DNA oxidizing agent that is widely used to detect local DNA melting. Treatment of naked probe 2 by OP-Cu or KMnO4 revealed a hypersensitive region around position 12 (Fig. 6, a, lane 2, and b, lane 5). This is because of the heteroduplex segment at 12/11 (for OP-Cu-mediated DNA cleavage) and to the unpaired thymine at position 11 (for KMnO4-mediated DNA cleavage) (Table Ia).
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54 InteractionsThe hypersensitive site at position 12 is evident in reactions containing only
54 and probe 2 (Fig. 6, a, lane 3, and b, lane 1). This is consistent with
54 being weakly bound to probe 2 in the absence of any nucleotide-bound forms of PspF (see above). Strikingly, in the presence of PspF1275-ATP
S or PspF1275-ATP/ATP
S the hypersensitivity site at 12 almost completely disappeared in the OP-Cu reactions, demonstrating altered interactions with the 12 promoter region (Fig. 6a, lanes 4 and 5). In contrast, the hypersensitive site at position 12 was still evident within the ATP
S and ATP
S/ATP complexes when probed with KMnO4 (Fig. 6b, lanes 2 and 3). Yet, the intensity of the cleavage at the 11 thymine is reduced in the ATP
S-complex when compared with the ATP
S/ATP-complex (Fig. 6b, compare lanes 2 and 3). This difference in cleavage intensity was also observed on the probe 2 version of the
54-dependent E. coli glnHp2 promoter (data not shown), and again indicates changed interactions with the 12 promoter region.
54-RNAP InteractionsAs before (Fig. 2), the binding of
54-RNAP to probe 2 did not affect the inherent hypersensitive site at the 12 position to cleavage by OP-Cu (Fig. 6a, lane 6). However, in the presence of PspF1275-ATP
S or PspF1275-ATP
S/ATP,
54-RNAP interactions with probe 2, particularly around the 12 position, are changed as demonstrated by the reduction of OP-Cu-mediated cleavage (Fig. 6a, compare lanes 3 and 6 with 4 and 5 and 7 and 8, respectively). The progressive increase in the cleavage intensity at 12 in the presence of PspF1275-ATP
S, PspF1275-ATP
S/ATP, and PspF1275-ATP, respectively (Fig. 6a, compare lanes 7, 8, and 9), supports the view that
54-RNAP interactions with the 12 promoter region are altered in response to interactions with certain nucleotide-bound forms of PspF.
To determine whether the DNA has locally melted in response to PspF1275-ATPS and PspF1275-ATP
S/ATP, we probed complexes formed between the
54-RNAP and probe 2 in the presence of PspF1275-ATP
S and PspF1275-ATP
S/ATP, respectively, by KMnO4. No local DNA melting toward the transcription start site was detected (data not shown). However, consistent with the OP-Cu footprinting data, binding of
54-RNAP to probe 2 in the absence of nucleotide-bound forms of PspF did not protect the thymine at 11 from oxidation by KMnO4 (data not shown). Furthermore, differences in the cleavage pattern at the 11 thymine were observed in the complexes formed between
54-RNAP and probe 2 in response to PspF1275-ATP
S and PspF1275-ATP
S/ATP on the probe 2 version of the S. meliloti nifH and E. coli glnHp2 promoters (data not shown). These differences were very similar to those seen with
54 (see above and Fig. 6b). Overall, the OP-Cu and KMnO4 footprinting data strongly suggest that different nucleotide-bound forms of PspF have different effects on the 12 promoter region interactions made by the
54-RNAP, but that local DNA melting does not occur.
Action of PspF1275-ATPS and PspF1275-ATP
S/ATP on
54 and
54-RNAP Leads to Altered Interactions with Promoter DNA
Next, we attempted to differentiate the interactions created between the promoter DNA and 54 or
54-RNAP in the PspF1275-ATP
S and PspF1275-ATP/ATP
S complexes with probe 2 by DNase I footprinting (Fig. 6c). Binding of
54 or
54-RNAP to the S. meliloti nifH homoduplex probe or probe 1 (Table Ia) protects the DNA between positions 34 and 5. These protections extend further in the downstream direction in open promoter complexes formed by the
54-RNAP on the homoduplex probe, indicating isomerization of the
54-RNAP in response to ATP hydrolysis by the activator (11, 14, 19).
54-RNAP InteractionsComplexes were formed between the
54-RNAP and probe 2 in the presence and absence of various nucleotide-bound forms of PspF. As expected, in the absence of PspF the DNA is protected between positions 34 and 1 by the
54-RNAP (Fig. 6c, lane 3). The presence of PspF1275-ATP
S leads to an extended footprint in the downstream direction (Fig. 6c, lane 4); indicating isomerization of the
54-RNAP-probe 2 complex in response to PspF1275-ATP
S. Interestingly, such an extension is not evident in the presence of PspF1275-ATP
S/ATP (Fig. 6c, compare lanes 4 and 5). In striking contrast to the extended footprint seen on the homoduplex probe, the
54-RNAP footprint on probe 2 appears to shorten in the upstream direction in response to the action of PspF1275-ATP (Fig. 6c, lane 6). It seems that ATP
S, ATP
S/ATP, and ATP each cause changes in
54-RNAP-promoter interactions.
54 InteractionsConsistent with the gel-mobility shift assays (Fig. 1), no strong interaction between
54 and probe 2 was detected in the absence of PspF1275. However, at higher concentrations of probe 2, a weak
54-probe 2 complex could be detected. Analysis of this complex by DNase I footprinting revealed that the
54 footprint was short (between 34 and 10; data not shown), in contrast to that seen with probe 1 (between 34 and 5; Refs. 11, 14, and 19). In the presence of PspF1275-ATP
S, the
54 footprint on probe 2 is extended in the downstream direction (beyond position 1) as seen with the
54-RNAP (Fig. 6c, compare lanes 7 and 4). Interestingly, in the presence of PspF1275-ATP
S/ATP,
54 footprints the DNA between 34 and 5, as seen in complexes formed between
54 and probe 1 or the homoduplex probe (Fig. 6c, lane 8 and data not shown).
Overall, these footprinting results suggest that different sets of activator-dependent DNA interactions (within and outside the 12 promoter region) are made by 54 and
54-RNAP depending on the nucleotide bound states of the activator and involves promoter sequences downstream of 12 to about 1 (the promoter region where DNA melting is seen in normal open complexes).
Role of 54 Region 1 in PspF1275-ATP
S and PspF1275-ATP
S/ATP-mediated Complex Formation
Previous data have indicated that region 1 of 54 and the promoter sequences at 12 are intimately involved in the activator responsiveness of the
54-RNAP-closed complex (7, 8, 11). The interaction
54 region 1 makes with the 12 promoter sequence generates a nucleoprotein target for the activator (12, 13). We investigated the role of
54 region 1 in PspF1275-ATP
S and PspF1275-ATP
S/ATP mediated binding of
54 to probe 2.
1
54The binding of wild-type
54 and a mutant form of
54 lacking its amino-terminal residues 156 (
1
54) to probe 2 were compared. In contrast to the wild-type
54,
1
54 bound the promoter probe 2 well (Fig. 7a, compare lane 2 and 6), suggesting that region 1 inhibits the initial binding activity of
54 to promoter probe 2. To examine whether formation of the ATP
S- and/or the ATP
S/ATP-complex requires
54 region 1, we added PspF1275-ATP
S or PspF1275-ATP
S/ATP to the
1
54-promoter probe 2 complex. As shown in Fig. 7a, no ATP
S-complex or ATP
S/ATP-complex was detected (compare lanes 3 and 7 and 4 and 8, respectively). Similarly, the addition of PspF1275-ADP·AlFx to the
1
54-probe 2 complex did not result in trapped complex formation (data not shown). Therefore, we conclude that the increased binding of
54 to probe 2 is likely to be because of PspF1275-ATP
S, PspF1275-ATP
S/ATP, or PspF1275-ADP·AlFx-mediated conformational change of
54 region 1. Overall, it appears that in the absence of activation or in the presence of sequences within the fork junction that prevent tight binding (as in probe 2), region 1 of
54 acts negatively so as to limit DNA binding.
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DNase I Footprints of the 1
54-Probe 2 ComplexWithin the ATP
S-complex, the
54-specific footprint is extended in the downstream direction (beyond position 1), contrasting conventional
54 footprints on the homoduplex probe or probe 1 where protection occurs between positions 34 and 5 (Fig. 6c, lane 7, and data not shown). To determine whether this extension is dependent on region 1 of
54, we conducted DNase I footprinting on the
1
54-probe 2 complex. The DNase I footprint of the
1
54-probe 2 complex was similar to the wild-type
54 footprint (protection between positions 34 and 8). However, this footprint was unchanged in the presence of PspF1275-ATP
S contrasting that seen with full-length
54 (data not shown). This indicates that
54 region 1 contributes to the extended protection of DNA in the ATP
S-complex.
Region 1 Ala MutantsBy using a series of triple alanine substitution mutants (Ala mutants) in 54 region 1 (10), we attempted to identify region 1 residues that (i) prevented binding of
54 to probe 2 and (ii) allowed ATP
S- and ATP
S/ATP-complex formation. Initially, we tested the binding activity of the Ala mutants to probe 2. In contrast to
1
54, none of the alanine mutations resulted in significantly increased binding of
54 to probe 2 (Fig. 7b). This suggests that several sequences in region 1 prevent initial binding of
54 to probe 2. Based on the ability of the Ala mutants to form the ATP
S-complex, two classes of mutants can be distinguished: Class 1 includes the Ala mutants 68, 911, 1214, 1517, 2123, 2426, 3032, 3335, 3638, 4244, 4547, and 4850 that did not show increased probe 2-binding activity in response to PspF1275-ATP
S (Fig. 7b). Class 2 essentially includes the wild-type and mutants 2729 and 3941 that show an increased probe 2-binding activity in response to PspF1275-ATP
S (Fig. 7b). Thus, the data suggest that only a small group of residues in region 1 of
54 are unimportant for productive interaction between
54 and PspF1275-ATP
S.
In contrast to PspF1275-ATPS, PspF1275-ATP
S/ATP greatly stimulated the probe 2-binding activity of the Ala mutants. As shown in Fig. 7c, a further two classes can be distinguished: Class 1 essentially includes the wild-type and all Ala mutants between residues 6 and 14 (Fig. 7c, 68, 911, and 1214) and 2750 (Fig. 7c, 2729, 3032, 3335, 3638, 3941, 4244, 4547, and 4850). Class 2 includes the Ala mutants between residues 15 and 26 (Fig. 7c, 1517, 1820, 2123, and 2426) that did not form the ATP
S/ATP-complex effectively. Strikingly, this ATP
S/ATP-complex formation pattern is consistent with previous observations in which the
54-RNAP formed with the Ala mutants (15, 16, 17, 18, 19, 20, 21, 22, 23, and 24, 25, 26) failed to efficiently catalyze transcription in vitro in response to subsaturating PspF concentrations (10). Given that the Ala 2123 mutant fails to stably bind the PspF1275-ADP·AlFx (13) and residue 20 (Gln-20) localizes proximal to the 12 promoter region within
54-RNAP closed complexes (19), it appears that residues 1526 are required for the PspF1275-ATP
S/ATP-dependent tight binding of
54 to probe 2. Importantly, the differences in probe 2-binding activity observed with the Ala mutants highlights the fact that different
54 region 1 residues are involved in interacting with different nucleotide-bound forms of the activator that consequently leads to different sets of promoter interactions.
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DISCUSSION |
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Mechanochemical Functions of PspFThe ways in which PspF uses ATP binding and hydrolysis to promote open complex formation by 54-RNAP are not well understood. The effects of ADP·AlFx, ATP
S, and ATP
S/ATP-bound forms of PspF have on
54-DNA interactions begin to address this issue. Several lines of evidence have shown that the
54-RNAP makes use of a fork junction DNA structure to limit DNA opening prior to activation (7, 8, 11). An extension of this view is that changed interactions at the fork junction are required to allow open complexes to form. Variations in promoter sequences suggest that a range of natural DNA fork junctions will exist, and that although we have used artificial fork junctions in our work, closely related structures will exist in many natural
54-dependent promoter complexes. The DNA band shift results establish that the functional state of the activator required for stable binding to the
54-RNAP was created by interaction with ADP·AlFx, an analogue of ATP at the transition state for hydrolysis. This stable binding of the activator to the
54-RNAP leads to altered interactions between
54 and the fork junction structure at 12. The amino-terminal region 1 of
54 is required for creating the fork junction structure and activator binds directly to region 1. This suggests that the activator is able to couple events in ATP hydrolysis to changes in interactions between
54 and promoter DNA through restructuring the
54 determinant, region 1, that creates and maintains the fork junction structure. This view is consistent with the chaperone-like property of AAA proteins and predicted changes in activator protomer structure during ATP binding and hydrolysis (5, 23). Our data indicate functional significance for different nucleotide bound states of PspF.
New Binary 54 and
54-RNAP Promoter ComplexesResults with ATP
S and ATP
S/ATP suggest that a set of interactions between the activator and
54 or
54-RNAP can occur in response to changes in the DNA binding properties of
54. Again, the critical property of the DNA probe used to demonstrate changes in DNA binding was the presence of a fork junction structure. In contrast to the results with ADP·AlFx, no stable ternary
54-DNA complex was seen with ATP
S or ATP
S/ATP and activator, suggesting that more than one nucleotide-bound form of activator can transiently interact with
54 and
54-RNAP to change its DNA binding properties. This ATP
S-dependent transient interaction is presumably not dependent upon rapid hydrolysis of the triphosphate, but could involve sensing of the
-phosphate prior to hydrolysis or the formation of some intermediate in a reaction related to normal ATP hydrolysis. Because the ADP·AlFx-bound form of the activator also changed DNA binding properties of the
54 and
54-RNAP at the same fork junction probe, it seems that activator bound by ATP
S, ATP
S/ATP, or ADP·AlFx have some shared functionality. It seems that ATP
S can cause the activator to overcome an energetically unfavorable interaction between
54 and the DNA fork junction that is otherwise inhibitory for
54-DNA binding interactions. The inhibition appears to be caused by region 1 of
54 and region 1 also seems to be needed for the formation of the ATP
S- and ATP
S/ATP-dependent binary complexes.
Nucleotide Hydrolysis-independent Isomerization of 54 There are remarkable contrasts to previous results in which region 1 of
54 was required to bind to a set of heteroduplex probes carrying certain 12 fork junction structures and where ATP hydrolysis was necessary to remodel and isomerize the complex (probe 1 and variants thereof: Refs. 11 and 14, and Table Ia). In this work
54 region 1 inhibits binding to the heteroduplexes with alternative fork junctions and poorly hydrolyzed NTPs are used to remodel the
54 without stable DNA melting (probe 2; Table Ia). DNA heteroduplexes with opposing requirements for ATP versus ATP
S for altered binding of
54 may reflect two alternate states of a natural promoter: one in which unfavorable interactions caused by region 1 of
54 need to be overcome for binding (e.g. on probe 2; Table Ia and this work), and one in which a strong initial set of interactions that rely upon region 1 need to be changed (e.g. on probe 1; Table Ia, and Refs. 11 and 14). The extent to which the proposed alternate states of the promoter contribute to activation is likely to be DNA sequence-specific, and therefore promoter-specific. Several lines of evidence indicate the ATP
S-dependent reaction relates closely to the normal activation of
54-dependent promoters. These are the common reliance upon (i) region 1 of
54, (ii) the integrity of the GAFTGA sequences in PspF, (iii) the "arginine" finger in PspF, and (iv) similar concentration dependences upon
54, PspF, and nucleotide.
Nucleotide-dependent Activation of TranscriptionBecause a common core RNAP is used by the enhancer-independent class of factors, it would appear that the special features of
54-dependent transcription relate closely to the activator targeting an unusually stable
54-RNAP-fork junction complex. Some of the
54-RNAP-DNA interactions that activator change seem to have a modest energetic cost, as demonstrated by the action of the ADP·AlFx- and ATP
S-bound forms of the activator. Others, notably the DNA opening per se seems to correlate to ATP hydrolysis and appear to have a higher energetic cost. A feature of AAA proteins is the use of certain amino acid sequences that function to "sense" the state and the presence of the
-phosphate of ATP, and potentially relay this information to cause conformational changes required for their biological output. The shared property of ADP·AlFx- and ATP
S-bound forms of the activator in causing changes in DNA binding by
54 and its RNAP may well be related to a
-phosphate sensing process upon binding to these ATP. The failure of ADP, ATP, and AMP-PNP to behave like ADP·AlFx and ATP
S can be explained by differences or absences of
-phosphate interactions among the various nucleotides tested. For ATP
S or ADP·AlFx the sensing of the
-phosphate of the ATP is implicated as critically changing the functionality of the activator, a common theme for AAA proteins where nucleotide binding and hydrolysis control the binding interactions needed for substrate remodeling. It seems that the nucleotide-dependent changes in
54 and DNA structure lead to the delivery of the promoter DNA into the DNA binding cleft of the RNAP where stable melting occurs.
OverviewOur results can be viewed as providing snapshots of potential intermediates in the activation process. The use of DNA probes that mimic the proposed states of promoter DNA, and the ATP analogues provide the useful tools. However, proof of the mechanism will require detailed time resolved analyses of ATP-dependent conformational changes in the native protein and DNA components. The effects of ATPS and ATP together upon the activity of PspF is interesting and suggests new or increased functionality through different bound nucleotides and creation of a mixed nucleotide bound state. Structures of the AAA proteins HslU and p97 have shown that binding of different nucleotides leads to conformational changes in HslU and p97 (24, 25). Here the content of the nucleotide binding pocket determines HslU conformation by bringing the
/
and
-helical domains of HslU together. In so doing, the I-domain of HslU is moved. The
54 binding site in PspF is believed to be equivalent to the I-domain of Hs1U (5, 17), and so the combined effects of ATP
S and ATP or ADP might be explained by their effects upon the
54 binding site in PspF. In relation to ATP hydrolysis, structural differences between the ATP and ADP bound states may be a key element in how PspF acts on
54 and its RNAP. Large conformational changes associated with ATP binding as opposed to hydrolysis are common in ATP-dependent molecular machines (25). It seems a range of nucleotide-bound protomers of PspF will contribute to full activation, some contributing more toward particular steps than others, and some preceding hydrolysis and product release. The precise nucleotide dependence seems to be DNA-dependent, indicating that energy coupling in
54-dependent systems critically involves key promoter sequences that communicate via the
factor to the activator. The interaction of
54 with the fork junction DNA is clearly subject to regulation and given the similarity between bacterial and eukaryotic multisubunit RNAPs, a range of control proteins may act to regulate transcription through targeting the fork junction structure. Interestingly TATA-binding protein DNA binding is regulated in an ATP-dependent fashion by Mot1 (26).
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FOOTNOTES |
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These authors contributed equally to this work.
To whom correspondence should be addressed. Tel.: 44-207-594-5442; Fax: 44-207-594-5419; E-mail: m.buck{at}imperial.ac.uk.
1 The abbreviations used are: RNAP, RNA polymerase; AAA, ATPase associated with various cellular activities; PspF, phage shock protein F; OP-Cu, ortho-copper phenanthroline; ATPS, adenosine 5'-(
-thio)triphosphate; AMP-PNP, adenosine 5'-(
,
-imido)triphosphate; GTP
S, guanosine 5'-(
-thio)triphosphate; ADP A1Fx, aluminum fluoride.
2 W. Cannon and M. Buck, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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