A novel bacterial transcription cycle involving 0genesdev.cshlp.org/content/9/18/2305.full.pdf0 -54 alone does not bind the glnAp2 promoter (Ninfa et al. 1987, and confirmed below)

A novel bacterial transcription cycle involving 0 .54 Yin Tintut, Jonathan T. Wang, and Jay D. Gralla 1

Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90024-1569 USA

0.54 is the promoter recognition subunit of the form of bacterial RNA polymerase that transcribes from promoters with enhancer elements. DNase footprinting experiments show that 0.s4 is attached selectively to the template strand, which must be single-stranded for transcription initiation. 0.54 remains bound at the promoter after core polymerase begins elongation, in contrast to the well-established 0.7~ transcription cycle. Permanganate footprinting experiments show that the bound 0 .54 and the elongating core RNA polymerase downstream of it are each associated with a single-stranded DNA region. Template commitment assays show that the promoter-bound 0.54 must be reconfigured before reinitiation of transcription can occur. This unexpected pathway raises interesting possibilities for transcriptional regulation, especially with regard to control at the level of reinitiation.

[Key Words: os4; footprinting; transcription cycle]

Received May 8, 1995; revised version accepted August 3, 1995.

o s4 is the only known o factor in Escherichia coli that is not a member of the o TM family of proteins (Merrick and Gibbons 1985; Lonetto et al. 1992). It mediates transcriptional responses to a variety of signals (for review, see Merrick 1993). These varied transcriptional responses are not united by a common physiological basis but are united by a common mechanism (for review, see Kustu et al. 1989; Collado-Vides et al. 1991). Although it uses the common core RNA polymerase, the o54-dependent mechanism differs from that exhibited by holoenzymes containing members of the o z~ family of proteins. Un- like the oZ~ system, all of the known o s4- dependent promoters are activated by enhancer-binding activators (Reitzer and Magasanik 1986; Collado-Vides et al. 1991), which are otherwise restricted to higher cells. Other features of the system also resemble mam- malian mechanisms, including the ability to form a sta- ble closed complex, the need to hydrolyze ATP to open the DNA, and the modular nature of the proteins in- volved in transcription (Gralla 1991; Wang et al. 1992; North et al. 1993). o s4 itself has three functional domains: The carboxyl terminus is required for the binding of promoter DNA; the amino-terminal region is required for activation; and the domain between these regions is for binding core RNA polymerase (Sasse-Dwight and Gralla 1990; Tintut et al. 1994; Wong et al. 1994).

The mechanism of activation at the glnAP2 promoter has been studied intensively both in vivo and in vitro (Ninfa et al. 1987; Sasse-Dwight and Gralla 1988;

1Corresponding author.

Popham et al. 1989); therefore, we used this promoter in our study. When sufficient nitrogen is present, oS4-holoenzyme forms a closed complex that occupies the glnAp2 promoter in an inactive state (Sasse-Dwight and Gralla 1988). When nitrogen becomes insufficient, a cas- cade of reactions occur leading to the phosphorylation of enhancer-binding protein NtrC, which then binds to a remote upstream sequence (Keener and Kustu 1988) and activates transcription. The activation event occurs via DNA looping (Su et al. 1990) and involves the use of ATP to convert the closed complex to an open complex, which is active for transcription (Ninfa et al. 1987; Sasse- Dwight and Gralla 1988; Popham et al. 1989; Weiss et al. 1991).

o s4 is unusual in that it binds to certain promoters such as Rhizhobium meliloti nifH (Rm nifHp) in vitro in the absence of core polymerase (Buck and Cannon 1992). This property of o s4 is intriguing because it is not shared by o TM (Dombroski et al. 1992, 1993). This unexpected property of o s4 raises the possibility that o54-holoen - zyme may engage in a transcription cycle that is different from that of o 7~ That is, the stronger contacts that bind o s4 to the promoter may be more difficult to break during transcription initiation. Therefore, instead of re- leasing a-factor from the promoter during transcription initiation, as in the case of o 7~ (Carpousis and Gralla 1985; Krummel and Chamberlin 1989), o s4 could conceivably be left behind at the promoter after the polymerase moves downstream for transcription. There is currently no evidence for this pathway in bacteria, but a similar pathway has been inferred to exist for mamma- lian transcription (Van Dyke et al. 1989; Jiang and Gralla 1993), which, as mentioned, shares features with o s4

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Tintut et al.

transcription. An initial goal of this study is to test whether 0-s4 is left behind at the glnAp2 promoter after transcription initiation in vitro.

We address whether 0-S4-holoenzyme uses a novel bacterial transcription initiation cycle using in vitro DNase I footprinting, KMnO4 probing, and template commitment assays. The results show that 0-s4 is left behind at the promoter during transcription. The mechanism and regulatory implications of the novel transcription cycle are discussed.

R e s u l t s

DNase footprints under steady-state transcription conditions

In these experiments we use primer extension procedures to footprint transcription complexes present on a supercoiled plasmid that contains the crS4-dependent promoter glnAp2. These procedures allow all footprinting experiments to be done on identical templates; different strands and regions may be probed by the use of different labeled oligonucleotide primers (Gralla 1985). The plasmid contains the activator (NtrC)-binding sites upstream of the basal promoter elements located near - 2 4 and - 12. When NtrC is phosphorylated it binds to its sites and activates 0-S4-dependent transcription.

The goal of the initial set of experiments is to determine whether there are any proteins bound at the glnAp2 promoter during ongoing transcription in vitro (see Ohlsen and Gralla 1992): This requires establishing conditions where the glnAp2 promoter is being actively used for transcription initiation and elongation and then applying DNase I footprinting to assay for protection of the basal promoter elements. To compare such results {see below) to data from known complexes that can form over the promoter, we first characterize closed and open complexes. These controls will be done in the absence of NTPs to accumulate these complexes. It is known that 0 -54 alone does not bind the glnAp2 promoter (Ninfa et al. 1987, and confirmed below). Closed complexes form when template DNA is incubated with purified core polymerase and 0-s4. To form an open complex, NtrC, the low molecular weight phosphate donor carbamyl phosphate (CBP), and ATP are added in addition to core and cr s4. CBP is included to phosphorylate NtrC (Feng et al. 1992). The polymerase, however, will not initiate transcription because the three other nucleoside triphosphates are absent.

Figure 1 shows the DNase I footprinting of the top strand of a supercoiled plasmid containing the glnAp2 promoter region. As shown previously, r alone does not bind to the glnAp2 promoter (lane 1 with 0-s4 vs. lane 2 without). When core RNA polymerase is also present, the closed complex that forms protects the DNA from - 3 4 to - 2 (lane 3 vs. lane 2). This complex is titrated away with heparin (lane 4), as expected for a closed complex (Popham et al. 1989). The footprint extends further to +23 when activator (NtrC-P) and ATP are present (lane 5) to form an open complex. This open complex is

Figure 1. DNase footprints of the top strand of the glnAp2 promoter. (Lane 1) 054; (lane 2): no 0"s4; (lane 3): closed complexes (CC) containing core polymerase and 0"s4; (lane 4): closed complexes treated with heparin (h) (10 ~g/ml); (lane 5): open complexes (OC); (lane 6): open complexes treated with heparin; (lane 7): open complexes treated with NTPs to allow transcription. The arrow indicates the + 1 start site and the direction of transcription. The four lanes at left are dideoxy chemical sequencing markers G, A, T, and C. The concentrations of proteins are NtrC (100 riM), 0"s4 (50 nM), core polymerase (25 nM), and the buffer A was used.

resistant to heparin, as expected (lane 6) (Popham et al. 1989).

Next we let the polymerase transcribe by adding the remaining ribonucleoside triphosphates to the open complex . The result shows that the footprint pattern changes when transcription is permitted. The pattern shows strong protection from - 34 to - 2, similar to that of a closed complex (lane 7 vs. lane 3), and partial protection in the downstream region protected in an open complex. The results indicate that under steady-state transcription conditions, the promoter site appears to contain a mixture of closed and open complexes. Because NTPs are present continuously, the experimental protocol cannot sort out what stage of RNA synthesis is associated with these complexes.

2306 GENES & DEVELOPMENT



~54 transcription cycle

DNase footprinting when reinitiation is blocked

The above experiments show that after NTPs are added to init iate transcription, the glnAp2 promoter still shows protected regions. Next we wish to learn whether these protected regions are a consequence of r being left behind. To do this, we repeat the above experiment but l imi t transcription to a single round. In this protocol, we add another r dependent promoter (Rm nifHp) after open complexes are formed at glnAp2 but before transcription has begun. The excess nifHp competitor binds any free RNA polymerase and thus prevents rebinding at the glnAp2 promoter. The control experiment confirms that competitor Rm nifHp DNA is in excess over glnAp2 {Fig. 2; lanes 4,5,7,12; no footprint when competitor is premixed wi th template).

Next we established a control to demonstrate that the polymerase leaves the glnAp2 promoter during initiation. The polymerase is expected to escape from the promoter and stall at position + 18 when RNA synthesis is

done in the presence of ATP, CTP, and GTP but not UTP. We provided this nucleotide combinat ion to trap stalled elongation complexes wi th competitor present to prevent formation of new complexes of the glnAp2 promoter. When the top strand of the glnAp2 was probed wi th DNase I under these conditions, the results show that footprint covers from +3 to +39 (Fig. 2B lane 6). This corresponds to the known footprint length of a r holoenzyme elongation complex (Carpousis and Gralla 1985) and confirms that in this experimental sys tem polymerase leaves the promoter during transcription and no new polymerase binds.

In a parallel experiment, also involving the use of competitor to prevent entry of new proteins, we add all four nucleotides to see any footprints that persist after transcription has begun. The sample was divided and probed wi th primers that detect modificat ions of either the top (as above) or bottom strands of DNA. When the top strand of the glnAP2 promoter was probed wi th DNase I, the footprint over the promoter disappeared (Fig. 2, A and

Figure 2. DNase footprinting when reinitiation is blocked. (A) Top strand. (Lane 1) closed complex (CC); (lanes 2,3) open complex (OC) with excess nifHp competitor added after open complex to prevent excess proteins from rebinding. NTPs were then added in lane 3 to allow transcription. (Lane 4) A control as in lane 2 except excess competitor was mixed with template prior to addition of proteins. (B) Top strand. (Lane 6) ATP, CTP, and GTP were added after the competitor to stall polymerase at position + 18 downstream; (lane 8) as in lane 6 except all NTPs were added; (lanes 5, 7) control patterns generated as in lane 4 of A. The start site and the direction of transcription is shown by an arrow and dideoxy sequencing markers for G, A, T, and C are displayed at left. (C) Bottom strand. (Lanes 9-12) As in lanes 1--4 of A showing closed, open, and transcribing complexes and control, respectively, except samples were probed on the bottom strand; (lane 13) DNA alone; (lane 14) DNA and r

GENES & DEVELOPMENT 2307



Tintut et al.

B, lanes 3,8), as expected from the above experiment, showing that polymerase has moved downstream.

However, the probing of the bottom strand of the same sample (Fig. 2C) shows a partial protection pattern corresponding to a protein that remains behind after the polymerase has left. Lane 11 has a banding pattern identical to the control (lane 12) above the position approxi- mately + 10. However, lane 11 shows partial protection below this position. This partial protection extends to cover at least the basal elements near - 24 and - 13 that ~s4 is known to bind at other promoters. The protection is not nearly as strong as that observed in an open complex containing r and core polymerase (lane 10). The protection, however, is much stronger than that seen when the top strand of the very same sample is probed (Fig. 2A lane 3 vs. lane 4, as discussed above). Thus, the protection pattern is strand specific. These results indicate that there is a transcription component, probably ~, left behind at the glnAp2 promoter after the first polymerase moves downstream for transcription.

Permanganate probing for single-stranded DNA

The DNase I footprints indicate that the factor left behind at the glnAp2 promoter site is bound primarily to one strand of DNA. Therefore, we used potassium permanganate to see whether the glnAp2 promoter is in a single-stranded state. Permanganate reacts preferentially with single-stranded thymines. Thus, it is often used to detect melted bubbles within transcription complexes and has been applied previously to the glnAP2 system (Sasse-Dwight and Gralla 1988). The permanganate patterns on the top strand of the glnAp2 promoter region are shown in Figure 3. The first two lanes are controls to show the patterns corresponding to closed (lane 1) and open {lane 2) complexes. Permanganate hypersensitive sites are present over the transcription start site in lane 2 (top bracket), as observed previously for open complexes. When cr s4 alone was incubated with the template, no permanganate-sensitive signal was detected (data not shown).

Next we probed the glnAp2 promoter with permanganate under conditions where polymerase is stalled at position + 18. The protocol allows the polymerase to initiate transcription and then elongate to this position but uses competitor nifHp to prevent any free proteins from associating with the promoter after the polymerase leaves (see above). The result confirms that a stalled elongation complex is formed, as evidenced by a new patch of permanganate hypersensitive sites at positions + 19, + 22, and + 23 (lane 3, bottom bracket). However, the pattern also shows a patch of hypersensitive sites remaining at the start site (top bracket). This pattern, showing two patches of hypersensitive sites, did not change with longer incubation times, consistent with the initiation reaction having reached an end point (data not shown). We infer that transcription to + 18 position is accompanied by two open regions, one at the site of stalled elongation and the other remaining over the transcription start site. Under the these same conditions,

Figure 3. KMnO4 probing for single-stranded regions. (Lane 1) Closed complex (CC); (lane 2} open complex (OC); (lane 3) polymerase stalled at the + 18 (see DNase footprint in Fig. 2B, lane 6); (lane 4) all four NTPs were added to allow transcription. The top bracket denotes the hypersites near the start site; the bottom bracket denotes hypersites at the + 19, + 22, and + 23 positions. Dideoxy sequencing markers are shown at left. The top strand was probed.

DNase protection was lost over the promoter site, which indicates that polymerase does not remain behind and that no new polymerase enters the glnAp2 promoter (Fig. 2B, lane 6). These results indicate that the start site remains single-stranded after polymerase leaves.

We compared this result to that obtained under transcription conditions where the DNase footprinting showed that a single strand-specific factor remains at the glnAp2 promoter (Fig. 2C, lane 11). Again excess nifHp competitor was present to prevent any reassociation of unbound proteins with glnAp2 after transcription had begun. The result shows that the glnAp2 promoter site remains sensitive to permanganate in the presence of four NTPs required for transcription (lane 4). This confirms that a factor, likely or, has been left behind in a single-stranded binding state at the glnAp2 promoter. Probing the opposite strand with K1VInO 4 showed properties consistent with these results (Tintut et al. 1995}.

Properties of the complex left behind

The DNase footprinting data indicate that the "left-behind" complex at the glnAp2 promoter does not contain polymerase in a conventional open complex. To compare this complex with the open complex we measured the lifetime of both. First, the lifetime of the open complex was measured. Open complex was formed on the glnAp2 promoter and competitor DNA was subsequently added




0-s4 transcription cycle

to titrate excess proteins and prevent rebinding. Then permanganate was added at different t imes to measure the amount of open complex remaining on the glnAp2 promoter (Fig. 4, lanes 1-5). The data show that there is only a small degree of dissociation during the 30-minute assay. Quant i ta t ive analysis of the data (not shown) indicates that the half-life of open complex is greater than an hour under these conditions.

To measure the half-life of the left-behind complex, we first formed an open complex on the glnAp2 pro' motet. Excess proteins were then titrated wi th competitor as described above. This was followed by the addition of all four NTPs, allowing the release of the first polymerase and leaving behind the complex to be assayed. Then the amount of complex remaining on the glnAp2 promoter was probed wi th permanganate at var- ious t imes (Fig. 4, lanes 6-10}. The data indicate that substantial dissociation of this complex has occurred during the 30-rain assay. Quanti ta t ive analysis indicates that the half-life of this complex is between 5 and 10 min. These data confirm that this complex is quite different from the open complex and is m u c h shorter lived, as expected for r lacking core polymerase.

Template commitment assay

Next we explore whether the left-behind r is functional in specifying transcription reinitiation. That is, we want to determine whether there is any kinetic advantage in attracting polymerase for successive rounds of transcription from the same template. We perform a template commi tmen t assay to address this question (see van Dyke et al. 1989).

The template c o m m i t m e n t assay measures whether a factor stays bound in a form that gives the template pref- erence for transcription. First, a l imi ted amount of holoenzyme is incubated wi th the original template for the init ial round of transcription. After the first round has begun, a second template is added to the mixture, and transcription including subsequent rounds is measured. In a case where r is funct ional ly commit ted to the original template, one wil l see exclusively transcription from the original template even though a second temo

Figure 4. Comparing the lifetimes of open and left-behind complexes. (Lanes 1-5) Open complexes were formed and then challenged with competitor for the indicated times before KMnO 4 probing. (Lanes 6--10) Left-behind complexes were formed by adding NTPs to open complexes. The samples were then probed with KMnO4 after the indicated times.

Figure 5. Template commitment transcription assay. (A) Tran- scription from the first template in the presence of different concentrations of 0 "54. (Lane 1) 10 riM; (lane 2) 20 riM; (lane 3)30 nM (lane 4) 40 riM. (B) Template commitment assay. (Lane 1) Reverse-transcribed RNA produced from a 20-rain reaction; (lane 2) identical, except the second template was added 1 rain after addition of NTPs; (lane 3) as in lane 1, except that rifampicin was added 1 rain after addition of NTPs. The arrow indicates the reverse-transcribed RNA produced from the original template. (C)) The reverse-transcribed RNA produced from the second template, which comigrates with the transcript produced from the upstream glnApl promoter.

plate is present. This is because ~ stays bound to the original template and in this protocol there is no free or; the original template is used in amounts known to be in excess over r We constructed two supercoiled templates both carrying the same glnAp2 promoter. The transcripts produced from these templates wil l have two different sizes as the original template contains a larger deletion wi th in the transcribed region.

First, we established conditions ensuring that the original template is in excess over ~. Figure 5A shows the amount of transcript produced from using different concentrations of cr s4, keeping the concentrations of all other proteins constant. As the r concentrat ion is increased from 10 to 20 to 30 nM, transcription increased (lanes 1-3). However, an increase in cr concentrat ion from 30 to 40 nM did not result in an increased amount of transcript (lane 3 vs. lane 4), indicating that r reaches functional excess only above 30 riM. Thus, we chose the

concentration of 20 nM to carry out the template com- m i t m e n t assay. The common steps in this assay are the formation of open complexes followed by the first round of transcription. The original template is incubated wi th core polymerase, as4, NtrC-P, and ATP for 20 min. Then nucleotides are added for 1 m i n for first-round transcription.

To determine the amount of RNA produced from the first round, NTPs were added, but reini t iat ion was




Tintut et aL

blocked by adding rifampicin. The amount of RNA produced 20 m i n later is shown in Figure 5B, lane 3 (arrow, 430 cpm in this example and 300 cpm in a second experiment). Second, we determine the amount of RNA produced in a parallel experiment that lacks r ifampicin (lane 1; 705 cpm in this example and 725 cpm in a second experiment). The quanti tat ion of the amounts of RNA in such experiments indicates that roughly two rounds of transcription occur wi th in this 20-min period.

Next we performed the template c o m m i t m e n t assay by adding a twofold excess of the second template to the reaction mixture after the common steps of allowing the original template to begin transcription. The results show that the amount of RNA from the original template is now restricted to an amount seen in a single round (Fig. 5B, cf. lane 2 wi th 455 cpm to lane 3 with 430 cpm, and in a second experiment cf. 300 cpm with 355 cpm). That is, the subsequent round of transcription from the first template is disrupted by the presence of the second template. This indicates that transcription is not funct ional ly commit ted to the first template after the first round, even though ~ is physical ly bound there. That is, the left-behind cr does not appear to collect new polymerase for transcription but, instead, slowly (tl/~ - 8 min) redistributes before the second round of transcription occurs. This is also evidenced by the appearance of some transcripts from the second template (Fig. 5B, lane 2, open circle). The inefficient transcription from the second template may be attributable to the slow off-rate of cr from the original template and the slow formation of open complexes on the second template.

This result raises the question of what is happening at the glnAp2 promoter after transcription initiation. The footprinting data indicate that cr s4 is left behind, but the template c o m m i t m e n t assay indicates that r cannot function in this state. We at tempt to observe this situation directly by allowing transcription ini t iat ion to begin and then adding r ifampicin to prevent reinitiation. No competitor is present, so under these circumstances the left-behind r has the opportunity to interact with free core polymerase. The state of the promoter is assayed wi th DNase and KMnO4 footprinting.

First, open complexes were formed on the glnAp2 promoter. Next nucleotides were added to allow first-round transcription. Rifampicin was then added to trap any complexes formed on the glnAp2 promoter after the first polymerase leaves. The top strand of the glnAp2 promoter was then probed wi th DNase I. The result (Fig. 6, lane 4) reveals a pattern that is unl ike open complex, although significant protection can be seen over regions protected in a closed complex (cf. to control lane 3 where competitor has been added to prevent any rebinding of core polymerase). Thus, at this t ime polymerase is not in the conformation ready to start the next round of transcription, even though the transcription start site was opened because of association wi th the left-behind r A parallel experiment using KMnO 4 probing confirms that the start site is open (lane 2) even though the new polymerase has the configuration on the DNA of a closed complex. Thus, the left-behind r keeps the DNA open,

Figure 6. Footprinting when reinitiation is blocked by rifampicin. Transcription was initiated with NTPs, and then rifampicin (0.125 mg/ml) was added to prevent a second round of transcription. (Lanes 2,4) KMnO4 and DNase footprints, respectively, after the addition of rifampicin; (lanes 1,3) as in lanes 2 and 4 except the competitor was added before NTPs to prevent association of new proteins with the left-behind ~s4. Buffer A was used. CC (closed complex) and OC (open complex) designate regions protected in those complexes, as determined in Figs. 1 and 2.

but when new core polymerase associates wi th the glnAp2 promoter, it cannot quickly assume the configuration associated wi th functional open complexes.

Discussion

Comparing the cr s4 and cr 7~ transcription cycles

The results demonstrate that cS4-dependent RNA synthesis at the glnAp2 promoter occurs using a novel transcription cycle. In this cycle r is left behind after core polymerase leaves downstream for elongation. DNase I footprinting shows that cr s4 holds on to the bot tom (template) strand of DNA, and permanganate probing confirms that the DNA start site remains open. The l i fet ime of r in this state is much shorter than that of the open complex, consistent wi th the lack of stabil ization from core polymerase. The selective interaction of cr s4 wi th the template strand is interesting in that it raises the possibility of a new role for cr factors; they may be the factor wi th in transcription complexes that holds the template strand in the appropriate position to allow the core polymerase to begin ini t iat ion at the correct nucleotide.




0 "s4 transcription cycle

The novel transcription cycle involving 0-s4 holoenzyme may seem unusual in view of the well-established and quite different 0-zo transcription cycle (see Krummel and Chamberlin 1989). However, the difference can be explained by the known differences between 0-s4 and 0-7o. One novel property of 0-s4 is the ability to bind to certain promoters alone, that is, without being part of a holoenzyme (Buck and Cannon 1992). Thus, this altered transcription cycle may arise from the difficulty in breaking tight contacts between 0-s4 and its primary DNA-binding determinant at - 2 4 (Wong et al. 1994), which remains double stranded during transcription initiation. 0-7o is not left behind after transcription begins, probably because it alone cannot bind to promoters (Dombroski et al. 1992 1993). 0-70 is lost in elongation complexes that contain transcripts as short as 10 or 11 nucleotides (Car- pousis and Gralla 1985; Krummel and Chamberlin 1989), in contrast to 0-s4 which remains DNA-bound even after polymerase has elongated a long RNA. Appar- ently, the ability of 0-s4 to bind DNA gives it the ability to participate in this altered transcription cycle.

Interestingly, the results from a template commitment assay of a 0-s4 promoter indicate that reinitiation from the original template is disrupted by adding an excess of a second template. This indicates that transcription is not committed to the original template after the first round of transcription even though 0-s4 remains bound. DNase I footprinting in the presence of rifampicin also shows that a new polymerase may enter but fails to protect the full promoter, indicating that it is in a conformation that is not competent to initiate transcription. Thus, the promoter cannot accommodate a new polymerase in a way that can engage immediately in reinitiation. This situation should not arise for 0-zo promoters, which should reinitiate transcription as soon as initiation has cleared the promoter (McClure 1985). Thus, the different transcription cycle involving 0-s4 gives it the potential to use different mechanisms for initiation and reinitiation.

quickly engage using a proper conformation, despite the fact that the start site is already open. Thus, although strong promoter contacts may cause 0-s4 to remain bound, this binding does not appear to facilitate assem- bly of new functional transcription complexes. We infer t h a t 0 -54 either needs to be released or undergo a confor- mational change prior to reinitiation. Either process could conceivably be facilitated by binding of new polymerase or by the state of activator, but this has not been tested.

As discussed above, this novel mechanism raises a new possible step at which transcription might be con- trolled, by allowing different mechanisms for initiation and reinitiation. In an in vivo setting, the bulk of RNA is produced not by the initial round of synthesis that follows induction, but by multiple rounds of reinitiation. Thus, one could control separately the rate of induction, via phosphorylation of activator, and the rate of reinitiation, via factors that work on the bound 0-s4 (see Jiang and Gralla 1993 for an analogous discussion of mamma- lian transcription). It is interesting to note that there are a group of open reading frames (ORFs) behind the 0-s4 gene on the chromosome that are cotranscribed with 0-s4 (for review, see Merrick 1993; Jones et al. 1994). It is possible that these ORFs or other unknown factors may participate in dissociating or reconfiguring 0-s4 at promoters to enhance or slow down the rate of transcription reinitiation, which makes the bulk of RNA from the promoter.

I NtrC- P ATP

~I osed complex

Mechanism and implications

The data suggest that transcription m vitro from the glnAp2 promoter begins as follows (see Fig. 7). The closed complex containing the holoenzyme forms first and is converted to an open complex by phosphorylated NtrC and ATP. In the presence of nucleotides, the polymerase then initiates transcription and moves downstream while 0-s4 is left behind at the promoter. This leads to templates containing two bubbles, one station- ary, associated with 0-, and the other moving downstream with core polymerase (Fig. 7). We have observed such a double bubble complex directly using permanganate probing (Fig. 3, lane 3). Preliminary experiments suggest that as initiation begins, the original bubble may become larger before separating into two smaller normal sized bubbles (Tintut et al. 1995).

After polymerase and 0- separate, the system has the potential to reinitiate. However, the data indicate that the new polymerase that comes in for reinitiation cannot

I NTPs

open compl e x

reinitiation pathways

el ongat i on cornpl ex

Figure 7. Model for the cr s4 transcription cycle. Under nonac- tivating conditions, holoenzyme occupies the promoter forming a closed complex. When enhancer protein NtrC is phosphorylated, it binds upstream and triggers open complex formation. After initiation, core polymerase and ~ separate; each remains associated with single-stranded DNA. Reinitiation requires release or reconfiguration of the bound (r.




Tintut et al.

These cons idera t ions raise n e w possibi l i t ies for tran- scr ip t ional control, at the level of re in i t ia t ion . In the in vi tro sys t ems cur ren t ly available, the rate of r e in i t i a t ion is predicted to be in f luenced by exper imen ta l condi t ions . These would inc lude the par t icular p romote r used because promoters vary in af f in i ty for cr s4. In addit ion, the s ta te of the t empla t e could be i m p o r t a n t (supercoiled D N A was used in th is study), as wel l as the t ranscr ip t ion buffer and sal t condi t ions . T h a t is, the abi l i ty of ~ to s tay beh ind and subsequen t ly be released would be expected to vary among expe r imen ta l sys tems, w i t h s trong binding promoters and supercoi led D N A possibly showing the greatest t e n d e n c y to ho ld r The poss ib i l i ty of chang- ing the factors and condi t ions of such exper imen t s should a l low fur ther tes t ing of models involv ing separate controls on i n i t i a t i on and re in i t ia t ion .

Mater ia l s and m e t h o d s

Materials

Supercoiled plasmid pLR1 which contains glutamine synthetase gene with glnAP2 promoter is a kind gift from Dr. B. Ma- gasanik (Reitzer and Magasanik 1986). Carbamyl phosphate, rifampicin, DNase I, and KMnO4 were purchased from Sigma, and NTPs were from Pharmacia. AMV reverse transcriptase, RNasin and Taq polymerase enzymes were from Promega. Pu- rified core RNA polymerase was purchased from Epicentre Technologies (Madison, WI), and Gr s4 and NtrC were purified as described (Popham et al. 1991; Reitzer and Magasanik 1983). Plasmid pJF5401 that overexpressed r was a kind gift from Dr. A. Ninfa (J.F. Feng, T.P Goss, R.A. Bender, and A.J. Ninfa, un- publ.), and the plasmid pCB5 that overexpressed NtrC is a kind gift from Dr. J. Moore (Moore et al. 1993).

Plasmids pLRl-dl and pLRlod2 are constructed as follows: pLR1 was linearized with the unique restriction enzyme BstBI, and nested internal deletions in the glnA gene were created with Bal 31. Competitor DNA was a 160-bp fragment carrying the Rm nifH promoter. Plasmid pYT1 was first constructed by li- gating a 55-bp fragment carrying the -45 to + 7 region of the Rm nifH promoter (Tintut et al. 1994) to pBR322 digested at a unique restriction site, EcoV. The competitor DNA fragment was generated by PCR amplification of pYT1 using primers flanking the Rm nifH promoter on the plasmid.

DNase I and KMnOa footprinting

Two buffer systems used in these experiments were buffer A (Tintut et al. 1994) [40 mM HEPES, at pH 8.0, 10 mM magnesium chloride, 100 mM potassium chloride, 1 mM dithiothreitol, 0.1 mM EDTA, 0.1 mg of bovine serum albumin per ml, 5% glyc- erol, 3.5% (wt/vol) polyethylene glycol (6000-8000; Sigma)] and buffer B (Buck and Cannon 1992) [25 mM Tris acetate (pH 8.0), 8 mM magnesium acetate, 10 mM potassium chloride, 1 mM dithiothreitol, 3.5% (wt/vol) polyethylene glycol]. Buffer B was primarily used except buffer A was used where indicated in some experiments. No differences in results were observed except slightly different KMnO4 patterns.

In the footprinting experiments the following common conditions were used. The core RNA polymerase (10 riM), cr s4 {20 riM), NtrC (40 riM), ATP (4 raM), carbamyl phosphate (10 mM) were incubated with template DNA pLR1 {0.5 nM) in buffer B (unless otherwise noted) for 20 min at 37~ Additions to the mixture, where indicated, were made in the following order.


Competitor DNA (48 riM) was added for 3 rain, followed by either CTP (0.5 mM) and GTP (0.5 raM) or all four ribonucle- otides (0.5 mM each) for 2 min, unless otherwise noted.

The samples were subjected to footprinting reagents as follows in a total of 40 ~1 : For DNase I digestion, 2 ~1 of DNase I (0.45 ~g/ml including 45 mM MgC12 and 22.5 mM CaC12) was added for 30 sec at 37~ The reaction was stopped with 2 ~1 of 0.5 M EDTA. For KMnO4 footprinting, 4 ~1 of 0.0925 M KMnO4 was added for 1 rain at 37~ The reaction was stopped with 6 ~1 of ~-marcaptoethanol. After treatment with footprinting reagents, 40 ~1 of phenol was added. The samples were then heated to 90~ for 4 rain, cooled, and centrifuged at 14,000g for 8 min. The aqueous layer was passed through a 1-ml syringe containing G50-80 equilibrated with water. The DNase I digested o r K M n O 4 modified template was then subjected to primer extension using 32P-labeled primers in PCR, as described previously (Sasse-Dwight and Gralla 1991). The PCR-extended products were separated on 6% denatured polyacrylamide gel.

Template commitment transcription assay

Transcription was carried out in a 40 ~1 reaction. The first DNA template (2.5 riM) was first incubated with purified proteins in 50 mM Tris-C1 (pH 7.8), 100 mM KC1, 10 mM MgC12, 0.1 mM EDTA, 1 mM DTT, and 50 ~g of BSA (Hunt and Magasanik 1985) in for 20 min. After a 1-min incubation with ribonucle- otides (0.5 mM each), either rifampicin (0.125 mg/ml) or a second DNA template (5 riM) was added. The incubation was con- tinued for an additional 20 min. Then the reaction was stopped by adding 40 ~1 of (5 M ammonium acetate, 100 mM EDTA) and 100 ~1 of ethanol. The DNA was precipitated, washed with 70% ethanol, and resuspended in 10 ~1 of reverse transcription mixture ( 1 x AMV reverse transcriptase buffer, 2 mM each dNTP, 20 units RNasin, 1 ~1 of labeled primer, and 4 units of AMV reverse transcriptase). The samples were incubated in 42~ for 1 hr, stopped by adding 10 ~1 of formamide dye, including 10 mM EDTA and 8 M urea, and separated on a 5% denatured polyacrylamide gel. For quantitation, phosphorimages of the gels were scanned using Molecular Dynamics (Sunnyvale, CA).

A c k n o w l e d g m e n t s

We thank Dr. A. Ninfa for providing ~54 expression vector, Dr. J. Moore for NtrC expression vector, and Z.S. Jwo for technical help. We also thank Dr. D. Nierlich, Dr. I. Gober, Dr. J. Moore, Dr. Y. Jiang, D. Mohl, and members of the Gralla laboratory for comments and discussions on the manuscript. This research was supported by U.S. Health and Human Services grant GM35754 to J.D.G., and by traineeship National Institutes of Health grant GM07185 to Y.T.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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10.1101/gad.9.18.2305Access the most recent version at doi: 9:1995, Genes Dev.

Y Tintut, J T Wang and J D Gralla A novel bacterial transcription cycle involving sigma 54.

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A novel bacterial transcription cycle involving 0genesdev.cshlp.org/content/9/18/2305.full.pdf0 -54 alone does not bind the glnAp2 promoter (Ninfa et al. 1987, and confirmed below)