(Received for publication, October 19, 1995; and in revised form, February 21, 1996)
From the
Repression of transcription of the abrB gene is essential to expression of many of the postexponential genes in Bacillus. The repression is due to the activity of the response regulator protein Spo0A. We have used in vitro transcription and DNase I and hydroxyl radical footprinting to explore the mechanism of transcription inhibition. Spo0A binds to specific DNA sequences (0A boxes), and two such boxes are found downstream of the tandem promoters for the abrB gene. The data indicate that both RNA polymerase and Spo0A bind simultaneously to a DNA fragment containing the promoters and the 0A boxes. The Spo0A prevents the polymerase from inducing DNA strand denaturation at the promoter for the abrB gene.
When Bacillus subtilis reaches the end of logarithmic growth, it enters a transition state in which cells express genes designed to search for alternative nutrient sources. These genes, such as proteases, amylases are under the direct control of a set of proteins termed transition state regulators(1, 2, 3) . One such regulator is the AbrB protein. AbrB has a positive effect on transcription of some genes, such as hpr(4) and represses transcription of genes such as tycA(5) , spoVG(6) , and aprE(2, 7, 8) .
The abrB gene is transcribed during vegetative growth, and during
transition and subsequently sporulation, its transcription is repressed
by the product of the spo0A gene(9) . The repression
is due to the binding of Spo0A protein to two consensus binding sites
(TGTCGAA, termed 0A boxes), which are located between positions
+11 and +17 and positions +21 and +27, relative to
the downstream (P2) of two reported transcription initiation start
sites (P1 and P2; (10) ). Spo0A is a member of the response
regulator class of proteins(11, 12) , and its activity
is modulated by phosphorylation (13, 14) . The
regulation of phosphorylation of Spo0A ties the level of Spo0A-P ()to multiple inputs reflecting a variety of aspects of cell
physiology(13, 15, 16) . Spo0A is both a
transcription repressor and a transcription activator, and in this
paper we address how Spo0A represses transcription.
The structure of the abrB promoter region suggested three possible models for Spo0A repression of abrB transcription. One model is the formation of a DNA loop between a Spo0A binding site that is upstream of the promoter (at position -214, relative to P2) and the Spo0A binding sites downstream of P2. The DNA loop would block RNA polymerase access to the two abrB promoters. In the second model, binding of Spo0A to the 0A boxes would exclude polymerase binding to the abrB promoter by steric hindrance. In the third model, both RNA polymerase and Spo0A bind simultaneously to the promoter region; however, the polymerase is prevented from initiating, or possibly elongating, RNA synthesis.
Figure 1: Structure of the plasmid pJM5134. pJM5134 contains an 814-bp DNA fragment that includes the abrB transcription initiation sites (P1, P2) and the two downstream and one upstream Spo0A binding sites (0A boxes). To test the effect of eliminating the upstream 0A box on transcription initiation, the template used was the Asp700 to EcoRI fragment. For most transcription inhibition assays, the DNA was cut at the EcoRI and HindIII sites. The fragment containing the promoter was isolated by electroelution from an acrylamide gel used to separate the fragment from the vector. For the structural studies, the DNA was labeled at the EcoRI site on either the template or coding strand before cutting with HindIII.
Labeled DNA fragments for protection assays were made by
first digesting pJM5134 with EcoRI. To label the coding
strand, the DNA was treated with the Klenow fragment of DNA polymerase
I in 1 Klenow buffer containing 35 µM dTTP, 50
µCi of [
-
P]dATP (DuPont NEN, 3000
Ci/mmol), and Klenow enzyme. The Klenow reaction was terminated, and
the fragment was released with HindIII. To label the template
strand, the EcoRI-cut DNA was treated with calf intestinal
alkaline phosphatase and then labeled with 0.6-0.8 mCi
[
-
P]ATP (ICN, 7000 Ci/mmol) using T4
polynucleotide kinase. After terminating the kinase reaction the
fragment was released with HindIII. The labeled fragments were
isolated by electrophoresis(19) . The position of the bases in
the footprints was determined using the end-labeled DNA fragments
treated with various restriction enzymes to generate specific size
markers.
RNA polymerase was isolated from Bacillus subtilis strain 168S(20) . The polymerase was greater than 95% pure and contained no DNase or RNase. Polymerase protein concentration was measured relative to bovine serum albumin using a modified Lowry assay(18) . The nucleotides used were FPLC-purified (Pharmacia).
Figure 5: DNase I treatment of DNA labeled on the coding strand. A, pJM5134 was cut with EcoRI and labeled with the Klenow fragment of DNA polymerase. The labeled fragment was released with HindIII, isolated, and used in DNase I protection assays. AG and AUG initiating conditions were tested. When present, the polymerase was 100 nM; Spo0A inputs were 100, 200, and 400 nM, or 400 nM when present alone, and the DNA concentration was 2 nM. Base pairs relative to the start site for transcription from P2 are indicated. The positions that are multiples of 10 are marked by short dashes, and intervening positions that are multiples of 5 are marked by dots. At the right can be seen one of the fragments used as a molecular weight marker for this gel, which runs at position +14. B, the autoradiogram in Fig. 5A was analyzed as described under ``Materials and Methods.'' The scans represent AUG initiating conditions containing the following: RNA polymerase alone (scan 1); RNA polymerase plus Spo0A (scan 2); RNA polymerase plus Spo0A-P (scan 3); Spo0A-P alone (scan 4); no protein (scan 5).
An example of the transcription inhibition by Spo0A and Spo0A-P is shown in Fig. 2. Transcription from both P1 and P2 was inhibited over a narrow range of Spo0A(-P) concentrations, and Spo0A-P was more efficient at inhibiting transcription (usually 2-5-fold). Transcription from both P1 and P2 was 90% inhibited by 400 nM Spo0A and 200 nM Spo0A-P. Order of addition experiments showed that under single round AG or AUG initiation conditions, the polymerase could be added before Spo0A or Spo0A-P and the response regulator could still inhibit transcription. This result was the first indication that the effect of Spo0A(-P) was not due to exclusion of polymerase binding. Five different preparations of recombinant Spo0A and two different preparations of RNA polymerase were tested, and all gave essentially identical results. Low inputs of Spo0A appeared to stimulate transcription slightly. The cause for this apparent stimulation is unknown.
Figure 2: Transcription inhibition by Spo0A(-P). The 814-bp EcoRI to HindIII fragment from pJM5134 was used as a template in a reaction that contained varying amounts of Spo0A (open squares) or Spo0A-P (open circles). The labeled transcripts were separated by electrophoresis. Gel slices containing the transcripts were localized by autoradiography and excised from the gel, and the level of product was determined by measuring the Cerenkov radiation in the gel slices. The data were normalized to the value in the absence of Spo0A(-P). In this experiment, the transcripts from P1 and P2 were summed. No difference in the response of the two promoters could be detected.
There is a Spo0A binding site 219 bp upstream of the abrB P2 promoter initiation site fragment, which might participate in DNA loop formation to inhibit abrB transcription (Fig. 1). We tested transcription of a template that had been shortened by cleavage with Asp700, removing the -219 Spo0A binding site. Transcription inhibition by Spo0A(-P) was unaffected by this change in template, indicating that the loop model did not explain Spo0A inhibition of abrB transcription (Fig. 3). We thus tested whether Spo0A prevented RNA polymerase binding to a DNA fragment containing the promoter.
Figure 3: Spo0A-P inhibition of Asp700-shortened template. The Asp700 to EcoRI DNA fragment was used as a template in transcription reactions with varying amounts of Spo0A-P. The radioactive transcripts were separated by electrophoresis, and the gel was exposed to x-ray film for 12 h.
Protection of the abrB promoter from DNase I digestion by Spo0A(-P) is shown in Fig. 4. The level of protection of the DNA was not linearly dependent on Spo0A(-P) input, and the two 0A boxes downstream of the promoter region appeared to fill simultaneously. Spo0A protected the abrB promoter at 400 nM but not 350 nM, while Spo0A-P protected the abrB template at 350 nM but not 300 nM. This behavior has been observed previously(10) . When the DNA was labeled at the 3` end of the coding strand, Spo0A protected DNA between positions +4 and +28, while Spo0A-P protection extended to -4 and +32. On the template strand, Spo0A provided weak protection between +6 and +28 even at high concentrations, while Spo0A-P protected positions between -3 and +32 (data not shown).
Figure 4: Spo0A(-P) protection of the abrB promoter region. pJM5134 was cut with EcoRI and labeled on the coding strand. The labeled fragment was isolated and mixed with either Spo0A or Spo0A-P and then treated with DNase I (4 µg/ml) for 10 s. The resulting fragments were separated by electrophoresis, and the gel was exposed to x-ray film. The amounts of Spo0A(-P) in the reactions were as follows: no Spo0A(-P) (lanes 1 and 18), 50 nM (lanes 2 and 10), 100 nM (lanes 3 and 11), 150 nM (lanes 4 and 12), 200 nM (lanes 4 and 13), 250 nM (lanes 5 and 14), 300 nM (lanes 6 and 15), 350 nM (lanes 8 and 16), 400 nM (lanes 9 and 17).
To examine the patterns of protection of Spo0A(-P) in the presence of RNA polymerase, mixtures of Spo0A(-P), RNA polymerase, and labeled DNA were treated with DNase I. Fig. 5A shows the DNase I digestion pattern obtained with DNA labeled at the 3` end of the coding strand. Spo0A-P protected positions -4 to +34 as seen in Fig. 4, and Spo0A was less effective at protecting DNA. Between positions -5 and -50, there was no effect of adding Spo0A(-P) on the DNase I digestion pattern.
Under AG initiation conditions (-UTP, Fig. 5A, left lanes), RNA polymerase alone protected several groups of bases. Positions -10 to -25 were well protected, while positions -14 to -23 were partially protected. Upstream of position -25, the promoter DNA contained regions that were inherently resistant to DNase I digestion; however, it was clear that the polymerase provided protection at positions -27, -28, -30, and -43 by the significant reductions in cleavage at these sites. DNase I cleavage at position -24 was enhanced by the presence of RNA polymerase. Under AUG initiating conditions (+UTP, right lanes), the DNase I digestion pattern observed when polymerase alone was added was similar to the pattern under AG initiating conditions, despite the fact that when UTP was included the polymerase could transcribe an RNA 13 bases long. A summary of the protection by Spo0A(-P) and by RNA polymerase alone is presented in Fig. 7.
Figure 7: Summary of the protection assays. Protection from DNase I and hydroxyl radical are shown in different lines. Positions are numbered relative to the start site for transcription from P2 (+1). Every 10th nucleotide on the coding strand is underlined. The -10 and -35 sequences for the P2 promoter are underlined with a heavier line. The 0A boxes downstream of P2 are enclosed. The bases protected by Spo0A(-P) are indicated by black dots on each strand. The smaller dots indicate less protection. Textured dots indicate bases protected by Spo0A-P but not Spo0A. Open bars indicate protection by RNA polymerase; dashed open bars indicate lower level protection by the polymerase. The patterns of protection from hydroxyl radical for the template strand are shown separately for AG and AUG conditions. The thin arrowheads indicate bases exposed to hydroxyl radical cleavage. The thicker solid arrowheads indicate bases exposed to DNase I by RNA polymerase, while the thicker striped arrowheads indicate bases exposed to DNase I by the presence of Spo0A.
To examine the question of exclusion, DNase I digestion reactions included RNA polymerase and either Spo0A or Spo0A-P. In all experiments, Spo0A(-P) was added and allowed to bind to the DNA first, and then polymerase was added and the mixture was subjected to a short DNase I treatment. Under AG initiating conditions, the effects of Spo0A differed from those of Spo0A-P. Spo0A addition increased protection at the -4 site and in the regions -27 to -33 and -37 to -44. At the same time, protection between +20 and +25 was slightly reduced. When Spo0A-P was added, the protection pattern between -35 and -43 and between -27 and -33 did not change relative to the pattern seen with polymerase alone. The addition of Spo0A-P increased protection between -14 and -20, while the protection between -1 and -12 decreased.
Fig. 5B shows scans of selected lanes from AUG initiating conditions in Fig. 5A. Comparison of the control (scan 5) and the Spo0A-P alone (scan 4) shows that between -4 and -50 the scans were identical, so that Spo0A-P had no effect on the DNase I digestion upstream of the -4 position. The presence of RNA polymerase alone (scan 1) dramatically altered the scan between positions +25 and -50. The peaks in the region -10 to +25 were significantly reduced. Other changes induced by the polymerase binding included the exposure of the base at -24 (increase in the peak height), the protection of the bases at -27 and -28 (disappearance of the peaks on the shoulder of the -24 peak), and the protection in the region of -43. When Spo0A and RNA polymerase were added together (scan 2), the pattern between -1 and -50 was essentially unchanged from that of the polymerase alone. The addition of Spo0A-P altered the pattern of protection seen with the polymerase, since the protection between -1 and -10 decreased slightly, although the polymerase was still clearly present, since these bases were not protected by Spo0A-P (scan 4). Furthermore, between -15 and -50, the protection by Spo0A-P plus polymerase was identical to that of the polymerase alone.
Several conclusions can be drawn from the data in Fig. 5. First, the areas of DNA protected by RNA polymerase alone, especially upstream of position -14, were not deprotected by the addition of Spo0A(-P). Thus, under conditions of complete transcription inhibition, it was evident that the polymerase was still bound to the promoter. If Spo0A-P blocked transcription by exclusion, the polymerase footprint should have been replaced by the Spo0A(-P) footprint. For example, the height of the -24 peak should have been reduced, and the peaks at -27 and -28 should have reappeared. Since the protection pattern seen with the polymerase was retained, we conclude that Spo0A(-P) did not exclude the polymerase from the template. Second, the addition of Spo0A-P caused some changes in areas of protection afforded by the polymerase alone. For example, adding Spo0A-P increased protection in the -14 to -20 region under AG initiating conditions and the -10 to -20 region under AUG initiating conditions. Since these nucleotides were not affected by binding of Spo0A(-P) alone, the changes can only be accounted for by having Spo0A-P and the polymerase bound to the same DNA. Finally, the interaction of Spo0A and Spo0A-P with the bound polymerase was not the same, although both inhibited transcription. The patterns of the protected bands in the -1 to -10 region (Fig. 5B, compare scans 2 and 3) are most illustrative of these differences. The significance of these changes is not clear as yet, but we presume they reflect differences in the structure of phosphorylated and unphosphorylated Spo0A.
An indication of the mechanism for Spo0A-P inhibition of abrB transcription was obtained by examining the hydroxyl radical cleavage pattern using DNA labeled at the 5` end of the template strand (Fig. 6). Spo0A-P protected two 3-bp areas (+8, +9, +10 and +18, +19, +20). Minor areas of protection were noted on either side, and Spo0A protected the same areas, although less effectively. Under AG initiating conditions (-UTP, right lanes) the polymerase alone induced strong hypersensitive sites between -5 and -2. Hypersensitive sites have been seen in other examples of hydroxyl radical treatment and have been interpreted as reflecting exposure of the DNA backbone due to DNA strand denaturation (see ``Discussion''). In addition, the polymerase protected several groups of bases spaced at nearly 10-bp intervals (see Fig. 7). Particularly noteworthy is the protection at -40 to -43. Under AUG initiation conditions, the hypersensitive sites shifted to +3 to +7, while the protection pattern between -10 and -45 was effectively identical to that seen under AG initiation conditions. Addition of Spo0A or Spo0A-P reduced or eliminated the hypersensitive sites under both AG and AUG initiation conditions, with Spo0A-P being more effective. In all tests, the addition of Spo0A(-P) had no obvious effect on the protection pattern between -10 and -45.
Figure 6: Hydroxyl radical treatment of DNA labeled on the template strand. pJM5134 was cut with EcoRI and labeled with the polynucleotide kinase. The labeled fragment was released with HindIII, isolated, and used in hydroxyl radical protection assays. AG and AUG initiating conditions were tested. When present, the polymerase was 100 nM; Spo0A inputs were 100, 200, and 400 nM, or 400 nM when present alone, and the DNA concentration was 2 nM. Base pairs relative to the start site for transcription from P2 are indicated. The positions that are multiples of 10 are marked by short dashes, and intervening positions that are multiples of 5 are marked by dots. At the right can be seen one of the fragments used as a molecular weight marker for this gel, which runs at position +16.
The change in hydroxyl radical hypersensitivity in Fig. 6is further evidence that the addition of Spo0A(-P) affected the interaction of the polymerase and the abrB promoter, since Spo0A(-P) itself had no effect on these positions. Furthermore, the change in the pattern in the presence of both Spo0A(-P) and RNA polymerase indicates that the patterns observed are not the summation of interactions of the polymerase and Spo0A(-P) with separate populations of DNA, since only by binding to the same DNA strands as the polymerase could Spo0A(-P) reduce the hypersensitivity to hydroxyl radical. Finally, the change in the hypersensitivity occurred without affecting the protection of the upstream regions of promoter by the polymerase, consistent with the interpretation that Spo0A(-P) did not displace the polymerase when it inhibited transcription at the abrB promoter.
The experiments we reported were designed to distinguish between three models for repression of abrB transcription by Spo0A. In the first model, Spo0A binding to a site at position -219, relative to the abrB P2 promoter and to the 0A boxes downstream of the P2 start site, would induce a formation of a DNA loop, which would inhibit transcription. Transcription regulation by DNA loop formation is well documented in Gram-negative bacteria(23, 24) . However, Spo0A inhibition of in vitro transcription of the abrB promoters was unchanged when the upstream site had been removed, arguing strongly against the loop model.
The second model predicted that Spo0A binding to the
downstream 0A boxes would exclude RNA polymerase binding to the
promoter region of abrB. The paradigmatic examples of this
model are the regulation of the P promoter of bacteriophage
by cI (25, 26) and the regulation of the trp promoter by the Trp repressor(27) . In the third
model, Spo0A would prevent transcription but would not exclude the RNA
polymerase from binding to the abrB promoter. Since the DNase
I and hydroxyl radical protection assays showed RNA polymerase
protection of the abrB promoter region in the presence of
Spo0A and Spo0A-P at concentrations that substantially inhibited
transcription, these experiments support the third model for Spo0A
repression of abrB transcription.
The hydroxyl radical
assays showed the template strand was hypersensitive to cleavage in the
presence of RNA polymerase. To explain the increased sensitivity of the abrB promoter, we propose that strand separation exposed the
sugar phosphate backbone of the template strand, increasing its
accessibility to the hydroxyl radical. The hypersensitive region
shifted as the predicted transcript length at abrB P2 changed
from 1 to 13 nucleotides, reflecting the expected movement of the
active site of RNA polymerase. We examined the hydroxyl radical
footprint at another promoter (from phage 29) and found both the
hypersensitivity and a change in the location of hypersensitive bases
when the polymerase could elongate RNA further (data not shown).
Similar sensitivity to hydroxyl radical attack was noted for E.
coli RNA polymerase at the T7 A1 promoter(28) , and the
hypersensitivity was also interpreted as reflecting exposure of the
template strand due to denaturation of the DNA. However, these
hypersensitive sites are not found at all promoters (for example, see (29) ). The hypersensitivity of the abrB promoter was
inhibited by the addition of Spo0A(-P), indicating that transcription
inhibition at the P2 promoter results from preventing the formation of
a complex with the DNA strands denatured. Since the assays we used
contained the initiating nucleotides, we cannot pinpoint more precisely
which step in initiation was blocked. In spite of the block to strand
separation, the hydroxyl radical assays showed that regions of the
promoter protected solely by the polymerase were still protected even
when Spo0A(-P) was present. This is consistent with the data from the
DNase I assays that Spo0A(-P) does not displace the polymerase from the abrB promoter. We presume that when a polymerase is bound at
P2 but cannot elongate RNA, it prevents transcription from P1, although
we have not directly tested this hypothesis.
One of the most extensively investigated transcription repressors is the lac repressor (LacI). While LacI has been shown to block initiation, conflicting findings about the precise step affected have been reported (see (30) and the references therein). A recent study by Schlax et al., carried out under conditions where formation of the initiated complexes was reversible, supported a model in which LacI and RNA polymerase compete for free promoter(30) . LacI appeared to block the formation of a complex with denatured DNA strands as we found for Spo0A(-P) inhibition at abrB P2, but for the lac case, there was no evidence for ternary complexes of repressor, DNA, and polymerase. The reactions conditions used by Schlax et al., are similar to the AG initiation conditions used in the work reported here in terms of concentration of polymerase, DNA, and repressor and the reversible state of the initiation complexes. In contrast, other studies, which have looked at the inhibition mechanism of LacI by examining stable initiation complexes, suggested that LacI and the polymerase were bound to the promoter simultaneously(31, 32, 33) . We would expect that the AUG initiation conditions at the abrB promoter would reflect a stable initiated state. While there were differences in the interaction of Spo0A(-P) and RNA polymerase, depending on the initiation conditions, the general finding that Spo0A(-P) inhibited transcription while binding along with the polymerase to the abrB promoter appeared to be independent of the reaction conditions.
Spo0A has been proposed to be a member of the helix-turn-helix family of DNA binding protein(35, 36) . The DNA binding by these proteins involves sequence-specific interactions by one helix (usually the C-terminal helix) and interactions with the phosphates in the minor groove by the other helix (reviewed in (37) ). The hydroxyl radical footprint thus indicates that the N-terminal part of the Spo0A helix-turn-helix region would make strong contact with the +7 to +10 region and +17 to +20 region of the abrB P2 promoter. This is compatible with proposed sequence-specific binding of Spo0A to the 0A boxes, which are between positions +11 and +17 and positions +21 and +27. Methylation protection experiments have shown that residues at positions +12, +20, +22, and +23 (on the coding strand) and positions +14 and +24 on the template strand are protected by Spo0A(10) . At higher inputs of Spo0A(-P), additional binding sites appeared in the hydroxyl radical footprint, separated by 10-bp intervals. These indicate that Spo0A may tend to ``line up'' on the DNA. While the physiological significance of this binding is not known, we believe it represents the tendency of Spo0A to self-associate on the DNA. Transcription and protection assays were sensitive to small changes in concentration of Spo0A(-P) (Fig. 2Fig. 3Fig. 4), and this sensitivity is probably necessary for Spo0A phosphorylation to have an effect on abrB transcription in vivo.
The appearance of hypersensitive sites in the DNase I assays (Fig. 4) indicated the Spo0A binding altered the conformation of the DNA particularly on the 5` side of the 0A box. Furthermore, the 5` boundary of the footprint with phosphorylated Spo0A was quite different from that seen with unphosphorylated Spo0A, a difference that may be due to changes in the structure of the protein that occurred upon phosphorylation.
Straney and Crothers (41) proposed a model for transcription initiation that included a stressed intermediate structure created by transcription that occurs before RNA polymerase releases upstream contacts at the promoter. E. coli RNA polymerase protected the lacL8 UV5 promoter from DNase I digestion between -54 and +23. Upon formation of abortive 6- or 8-mer transcripts, protection extended to +26 without RNA polymerase shifting from its interactions with upstream DNA. The upstream boundary for RNA polymerase protection of the abrB promoter region from DNase I remained unchanged during transcript elongation of 1 or 13 nucleotides, while the downstream protected region moved (Fig. 5Fig. 6Fig. 7). These results are similar to those obtained by Straney and Crothers (41) for the stressed intermediate complex formed at lacL8 UV5, suggesting that a similar stressed intermediate complex may form at abrB.