The Transcriptional Activator Protein FIS: DNA Interactions and Cooperative Interactions with RNA Polymerase at theEscherichia coli rrnBP1 Promoter

J. Mol. Biol. (1995) 245, 197–207

The Transcriptional Activator Protein FIS: DNA Interactions and Cooperative Interactions with RNA Polymerase at the Escherichia coli rrnB P1 Promoter Anton J. Bokal IV, Wilma Ross and Richard L. Gourse* Department of Bacteriology University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, U.S.A.

*Corresponding author

The E. coli rrnB P1 promoter owes its strength, in part, to the transcriptional activator protein FIS. FIS binds to three sites upstream of the RNA polymerase (RNAP) binding site and increases transcription in vivo four to ten-fold. In this report, hydroxyl radical and DMS footprinting analyses show that FIS binds to its three sites along one side of the DNA helix, and that FIS bound at the promoter-proximal site (site I) and RNAP bound at the promoter are in close proximity. The binding of FIS at site I and RNAP at the promoter are mutually cooperative. These observations support a model for direct interaction between the FIS protein bound at site I and RNAP in transcription activation at rrnB P1. We also find that FIS does not bind cooperatively to its three sites upstream of rrnB P1, and that the relatively small activation associated with FIS bound at sites II and III does not result indirectly by facilitation of binding of FIS to site I. Keywords: E. coli RNA polymerase; FIS protein; rRNA promoter; protein–protein interactions; transcription

Introduction The P1 promoter of the ribosomal RNA operon rrnB is one of seven Escherichia coli rrn P1 promoters which collectively account for the majority of RNA synthesis in rapidly dividing cells (Bremer & Dennis, 1987). DNA upstream of the − 10 and − 35 recognition hexamers for RNA polymerase (RNAP) is an important determinant of rrn P1 promoter strength (rrnA: Nachaliel et al., 1989; rrnB: Gourse et al., 1986; Zacharias et al., 1991; rrnD: Sander et al., 1993). The DNA upstream of rrnB P1 and rrnD P1 (upstream activating regions or UARs) has been dissected into two functionally distinct components (Ross et al., 1990; Leirmo & Gourse, 1991; Zacharias et al., 1992; Sander et al., 1993; Rao et al., 1994). The rrnB P1 sequence between − 40 and − 60, the A + T-rich UP element (Figure 1), interacts directly with the a subunit of RNAP and increases transcription at least 30-fold (Ross et al., 1993; Rao et al., 1994). The second component of the rrnB P1 UAR extends from − 60 to − 154, binds the FIS protein at three sites (Figure 1), and increases transcription an additional four to ten-fold (Ross et al., 1990). Since deletion of sites II and III results in only a 20 to 30% reduction of promoter activity

Abbreviations used: RNAP, RNA polymerase; DMS, dimethyl sulfate; CTD, C-terminal domain. 0022–2836/95/030197–11 $08.00/0

in vivo (Ross et al., 1990), FIS appears to activate rrnB P1 principally through site I. FIS is a site-specific DNA binding protein which participates in a variety of processes in addition to transcription activation of stable RNA promoters (Ross et al., 1990; Nilsson et al., 1990), including DNA inversion (Johnson & Simon, 1985; Kahmann et al., 1985; Johnson et al., 1986; Koch & Kahmann, 1986; Haffter & Bickle, 1987), phage l excision (Thompson et al., 1987; Ball & Johnson, 1991), and initiation of DNA replication at oriC (Gille et al., 1991; Filutowicz et al., 1992). FIS is a homodimer of 11.2 kDa subunits and its crystal structure has been determined (Kostrewa et al., 1991; Yuan et al., 1991). FIS binds DNA through helix-turn-helix motifs in the C-terminal regions of its subunits (Osuna et al., 1991; Koch et al., 1991) and recognizes a 15 base-pair (bp) degenerate consensus sequence (Hubner & Arber, 1989; Finkel & Johnson, 1992). The 40 to 90° DNA bend induced upon FIS binding may be influenced by sequences flanking the target sites (Thompson & Landy, 1988; Gille et al., 1991; Finkel & Johnson, 1992; Pan et al., 1994). Prokaryotic transcription activators can be distinguished by the locations of their binding sites with respect to the RNAP recognition hexamers, by the region of RNAP required for activation, and by their mechanisms of action (i.e. whether they increase transcription directly by interacting with RNAP or indirectly by altering DNA structure or facilitating 7 1995 Academic Press Limited

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Figure 1. Organization of the E. coli rrnB P1 promoter. rrnB P1 is composed of the − 10 and − 35 recognition hexamers for RNAP (small shaded squares), and the upstream activating region (UAR). The UAR consists of the UP element (hatched rectangle) and three FIS binding sites (open rectangles). + 1 is the transcription start site.

binding of other factors). Recent studies have shown that many activators interact with the C-terminal region of the a subunit of RNAP (class I activators; Ishihama, 1993; Ebright, 1993), and most of these bind upstream of the − 35 RNAP recognition hexamer. Although FIS binds upstream of the − 35 hexamer at rrnB P1, and there is suggestive evidence that it interacts with RNAP (Newlands et al., 1992; Gosink et al., 1993), its function does not require the C-terminal region of a (Ross et al., 1993). It is therefore designated a ’’non-class I activator’’ (Ishihama, 1993). We investigated FIS–DNA and FIS–RNAP interactions at rrnB P1 in order to clarify the mechanism of activation of rRNA transcription by FIS and to provide insight into the molecular architecture of a non-class I transcription activation complex. In this report we: (1) probe the interactions of FIS with its binding sites in the rrnB P1 UAR using footprinting techniques which reveal where it makes close approaches to the DNA; (2) test whether the binding of FIS at site I and the binding of RNAP at the promoter are mutually cooperative, a prediction of the ’’direct contact’’ model for FIS-mediated activation; and (3) investigate the mechanism by which sites II and III contribute to activation of rrnB P1.

Results

Mechanism of Activation of rRNA Transcription by FIS

recognition sequence, and two in the flanking regions (Figures 2A, 3). The positions protected on the two DNA strands in each segment are offset by 2–3 bp in the 3' direction, reflecting protein–DNA interactions situated across the minor groove from each other, and the centers of the protected segments are in phase with the helical repeat. Protection of the promoter-distal region of site III and the promoterproximal region of site I was either very slight or not detectable (Figures 2A, 3, 4 and data not shown). The presence of RNAP at rrnB P1 caused the promoter-proximal region of site I (arrow in Figure 4) to be more completely protected, but did not otherwise affect the interactions of FIS with its binding sites in the UAR (Figure 4 and data not shown). DMS methylates N-7 positions of guanine residues in the major groove, and bound proteins can block or enhance this methylation (Gilbert et al., 1976). Several positions in each FIS site reproducibly showed altered reactivity to DMS in the presence of FIS (Figures 2B, 3; positions − 64, − 65, − 66, − 67, − 77 and − 78 in site I; − 95, − 96, − 97, − 108 and − 109 in site II; and − 136, − 137, − 138, − 139, − 147 and − 150 in site III). The sites of altered DMS reactivity generally occur in analogous positions in the three FIS recognition sequences in rrnB P1 (Figure 3) as well as in the Hin enhancer (Bruist et al., 1987), indicating that FIS interacts similarly with these sites. When the rrnB P1 UAR was modelled as a linear B-DNA duplex with a 10.5 bp helical repeat, the . FIS-DNA interactions defined by the OH and DMS footprints map to one face of the DNA (Figure 5). Positions of altered DMS reactivity occur in two successive major grooves, each bounded by the . backbone positions protected against OH cleavage. RNAP protects two 4 to 5 bp segments within the upstream section of the RNAP binding site from . OH, centered at approximately − 42 and − 52 (Newlands et al., 1991). The narrow separation between the DNA surfaces in site I contacted by FIS and those in the UP element contacted by RNAP (Figure 5) suggest that the two proteins are in close proximity in this region.

Interactions of FIS with its binding sites in the rrnB P1 UAR The three FIS binding sites upstream of rrnB P1 were initially defined by DNase I footprinting (Ross et al., 1990). In order to localize more precisely the site-bound proteins on the DNA surface, we . determined the hydroxyl radical ( OH) and dimethyl sulfate (DMS) reactivities of the rrnB P1 UAR in vitro . in the presence or absence of FIS. The OH molecule cleaves DNA by abstracting hydrogen atoms from the deoxyribose sugars of the backbone (Hertzberg & Dervan, 1984). DNA backbone positions closely approached by site-bound protein are . not efficiently cleaved by OH (Tullius & Dombroski, 1986). FIS protected three 2 to 5 bp segments of . the DNA backbone from OH in each binding site: one centrally located within the 15 bp FIS

Mutually cooperative binding of FIS and RNAP Protein-protein contacts between FIS at site I and RNAP would be expected to facilitate the interaction of both proteins with their respective binding sites. In order to test this prediction in vitro, we used quantitative DNase I footprint titration to measure the affinity of FIS for site I in the presence and absence of RNAP and the affinity of RNAP for rrnB P1 in the presence and absence of FIS. To simplify the analysis, we used constructs containing only site I, since this site is responsible for the majority of FIS-dependent activation (Ross et al., 1990; Gosink et al., 1993). Briefly, end-labeled DNA fragments which contained site I and rrnB P1 were equilibrated with varying amounts of either protein in the

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Figure 2. Hydroxyl radical (A) and dimethyl sulfate (B) footprints of FIS bound at sites I, II and III in the rrnB P1 UAR. . DNA templates containing sites I, II and III were incubated with FIS for 10 min and then subjected to OH cleavage or DMS treatment as described (Newlands et al., 1991). Vertical lines indicate the extents of the FIS binding sites. Arrows in B indicate guanine residues which reproducibly exhibited altered reactivity toward DMS in the presence of FIS. A + G, G: DNA sequence markers. Complexes were prepared at 22°C in 150 mM NaCl, 10 mM Tris-HCl (pH 8), 10 mM MgCl2 , 1 mM DTT, and 100 mg/ml BSA. The FIS concentration was 45 nM (A), 180 nM (B, top strand) or 360 nM (B, bottom strand). The DNA templates were (BglI( − 299) to BamHI( − 28)) from pJNBend130 32P-labeled in the top strand at − 28 (A), [BamHI( − 160) to HindIII( + 50)] from pSL7 32P-labeled in the bottom strand at − 160 (A), [XhoI( − 168) to HindIII( + 50)] from pSL7 32P-labeled in the top strand at + 50 (B), and [BamHI( − 160) to XhoI( + 100)] from pSL7 32P-labeled in the bottom strand at − 160 (B).

presence and absence of the other and probed with DNase I. In reactions containing both proteins, one was included at a fixed concentration sufficient to saturate its target site. We generated binding isotherms by monitoring the presence of the titrating protein at its binding site, and determined equilibrium dissociation constants from these curves.

Effect of promoter-bound RNAP on the affinity of FIS for site I Several positions in site I were protected by FIS from DNase I. These positions became less accessible

to DNase I as the amount of FIS in the reactions increased (Figure 6A, B), reflecting increased site I occupancy. (Two positions within FIS site I are hypersensitive to DNase I in the presence of FIS (Figure 6; Ross et al., 1990). These positions map to the unbound face of the DNA (data not shown).) When RNAP was bound at rrnB P1, less FIS was required to protect site I from DNase I (e.g. compare lane 7 in Figure 6A with lane 7 in Figure 6B, reactions which contained equal amounts of FIS). In quantitative terms, the equilibrium dissociation constant for FIS binding at site I was reduced 3.4-fold, from 9.3 × 10 − 10 M to 2.7 × 10 − 10 M, when RNAP was bound at rrnB P1 (Figure 6C).

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Figure 3. Positions in sites I, II and III which exhibit . altered OH or DMS reactivity in the presence of FIS. Sequences are aligned with respect to the FIS consensus recognition sequence (Finkel & Johnson, . 1992). Positions (from Figure 2A) protected from OH attack are overlined (top strand) or underlined (bottom strand). Positions (from Figure 2B) which exhibited reduced (circles) or enhanced (carats) DMS reactivity are indicated.

Effect of site I-bound FIS on the affinity of RNAP for rrnB P1 DNase I signals in the promoter region and UP element decreased in intensity as the amount of RNAP in the reactions increased (Figure 7A, B), reflecting increased promoter occupancy. Less RNAP was required to protect rrnB P1 from DNase I when FIS was bound at site I (e.g. compare lane 5 in Figure 7A to lane 3 in Figure 7B, reactions which contained equal amounts of RNAP). In quantitative terms, the equilibrium dissociation constant for RNAP binding rrnB P1 was reduced 2.5-fold, from 7.6 × 10 − 9 M to 3.1 × 10 − 9 M, when FIS was bound at site I (Figure 7C). The initial binding step in the promoter–RNAP interaction is followed by a series of isomerizations which result in displacement of the leading edge of RNAP downstream of the transcription start site and separation of the DNA strands in the − 10 region of the promoter. Isomerization is inhibited by low temperature (Hofer et al., 1985; Kovacic, 1987; Cowing et al., 1989; Schickor et al., 1990). In order to measure the effect of FIS on the initial RNAP–rrnB P1 binding equilibrium in the absence of subsequent isomerization steps, the titrations of rrnB P1 with RNAP were done at 12°C (Figure 7). We verified in three ways that the initial RNAP–rrnB P1 complex did not isomerize under the conditions used in these titrations. First, positions downstream of the transcription start site were not protected from DNase I by RNAP irrespective of the presence of FIS (Figure 7A, B). Second, the binding of RNAP at 12°C did not increase the reactivity of rrnB P1 to potassium permanganate (KMnO4 ) in the absence or presence of FIS (data not shown). (KMnO4 preferentially oxidizes unpaired thymidine and

Figure 4. Hydroxyl radical footprints of FIS–site I complexes in the presence and absence of RNAP. DNA templates containing rrnB P1 sequences − 154 to + 50 were incubated with FIS for 10 min in the presence or absence . of RNAP and then subjected to OH cleavage as described (Newlands et al., 1991). The arrow indicates the position in site I which was more completely protected by FIS from . OH attack when RNAP was also present in the reaction (compare lanes 3 and 7 or lanes 4 and 8). A + G, G: DNA sequence markers. Complexes were prepared at 22°C in 30 mM KCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2 , 1 mM DTT, 100 mg/ml BSA, 500 mM ATP and 50 mM CTP. The FIS concentration was 24 nM (lanes 3 and 7) or 12 nM (lanes 4 and 8). RNAP was included at 10 nM where indicated. The DNA template, [BamHI( − 160) to XhoI( + 100)] from pSL7, was 32P-labeled in the bottom strand at − 160.

cytidine residues (Hayatsu & Ukita, 1967), and has been utilized often as a probe for strand separation in RNAP–promoter complexes (Sasse-Dwight & Gralla, 1989; Newlands et al., 1991).) Third, the initiating nucleotides, ATP and CTP, which induce strand separation or prevent strand reclosure in RNAP–rrnB P1 complexes at higher temperatures (Newlands et al., 1991; Ohlsen & Gralla, 1992), did not do so at 12°C (data not shown). Thus, by these three criteria the RNAP–rrnB P1 complex did not isomerize in our titration experiments (Figure 7), and

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. Figure 5. Space-filling model illustrating the OH and DMS reactivities of positions in the rrnB P1 UAR in the presence of FIS and RNAP. The rrnB P1 sequence from − 39 to − 154 was modelled as a linear B-form DNA duplex with a helical repeat of 10.5 bp/turn using Insight II software (BioSym Technologies). The 2 views of the DNA differ by a 180° rotation . about the helical axis. Backbone positions shielded from OH by FIS (Figure 2A) and by RNAP (Newlands et al., 1991) are dark gray. N-7 atoms of guanine residues differentially reactive toward DMS in the absence and presence of FIS (Figure 2B) are black.

FIS must, therefore, facilitate the initial RNAP–rrnB P1 interaction. Mechanism of activation by FIS bound at sites II and III Promoter–lacZ fusions with different rrnB upstream sequence boundaries showed that deletion of sites II and III results in a 20 to 30% reduction of

promoter activity in vivo (Ross et al., 1990). We asked if this small effect of sites II and III on activation of rrnB P1 still occurs even when site I is not bound by FIS, or if sites II and III activate transcription indirectly by facilitating the binding of FIS at site I. An rrnB P1 promoter with a mutation at position − 72 (D − 72) was used to ask whether FIS can activate rrnB P1 from sites II and III when site I is not bound by FIS. The D − 72 mutation abolishes FIS

Figure 6. DNase I footprint titration of site I with FIS in the presence and absence of RNAP. A, Equilibrium mixtures containing a DNA template with rrnB P1 sequences − 88 to + 50 were incubated with 40 nM RNAP for 10 min, and then varying amounts of FIS were added for 20 min before treatment with 4 mg/ml DNase for 30 s. B, In parallel reactions, the DNA was incubated with FIS in the absence of RNAP. Reactions in lanes 1 to 10 in A and B contained FIS at 0 nM, 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, 0.313 nM, 0.156 nM or 0.008 nM, respectively. Diagnostic and reference signals used for quantitation (see Materials and Methods) are indicated by filled and hollow arrows, respectively. Reactions were performed at 22°C in 120 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2 , 1 mM DTT and 100 mg/ml BSA. The DNA template, [XhoI( − 168) to NheI( + 75)] from pSL9, was 32P-labeled in the bottom strand at − 168. C, binding isotherms derived from the titration experiments shown in A (triangles; + RNAP) and panel B (circles; − RNAP).

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Figure 7. DNase I footprint titration of the rrnB P1 promoter with RNAP in the presence and absence of FIS. A, Equilibrium mixtures containing a DNA template with rrnB P1 sequences − 88 to + 50 were incubated with varying amounts of RNAP for 20 min and probed with DNase I (12 mg/ml) for 30 s at 12°C in the buffer described in Figure 6. B, In parallel reactions, the DNA was incubated with FIS at 2 nM for 10 min prior to addition of RNAP. Reactions in lanes 1 to 9 in A contained RNAP at 0 nM, 0 nM, 40 nM, 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM or 0.625 nM, respectively. Reactions in lanes 1 to 8 in B contained RNAP at 0 nM, 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM or 0.313 nM, respectively. Diagnostic and reference signals used for quantitation are indicated by filled and hollow arrows, respectively. The DNA template, [SmaI( − 138) to XhoI( + 100)] from pSL9, was 32P-labeled in the top strand at + 100. C, binding isotherms derived from the titration experiments shown in A (circles; − FIS) and B (triangles; + FIS).

binding and activation by site I (Ross et al., 1990). Footprints performed in vitro and in vivo confirmed that FIS does not bind to site I containing the D − 72 mutation even when sites II and III are occupied by FIS (W. Ross, A. J. Bokal, J. Salomon & R. L. Gourse, unpublished results). As expected, the D − 72 mutation decreased promoter activity in a fis + strain but not in a fis − strain (Figure 8), confirming that the D − 72 mutation affects only FIS binding at site I and not intrinsic promoter activity. The D − 72 promoter derivative containing sites II and III was 1.7-fold more active than the D − 72 promoter derivative lacking these sites in a fis + but not in a fis − strain (Figure 8, B versus A), indicating that sites II and III increase transcription independent of FIS binding at site I and that this stimulation is caused by FIS and not by the site II and III DNA sequence per se. However, sites II and III cannot compensate entirely for the loss of site I (Figure 8, B versus D). To confirm that sites II and III work independently and do not promote the binding of FIS at site I, we measured the affinity of FIS for site I in vitro on fragments containing or lacking sites II and III (Figure 9). Just as much FIS was required to protect site I from DNase I when sites II and III were also

present on the fragment (Figure 9). If anything, the presence of sites II and III slightly reduced the affinity of FIS for site I, increasing the equilibrium dissociation constant from 5.0 × 10 − 10 M to 7.7 × 10 − 10 M (Figure 9C).

Discussion FIS–DNA interaction . Our OH and DMS footprinting analyses indicate that the interactions of FIS with the DNA backbone and the major groove in each of the three FIS binding sites in the rrnB P1 UAR are confined to one face of the DNA (Figure 5). The pattern of protection by FIS against hydroxyl radical cleavage is very similar to that of a prototypic helix-turn-helix DNA binding protein, l repressor, with its operator, OR1 (Tullius & Dombroski, 1986). These observations are consistent with the model for FIS–DNA interactions derived from the studies of FIS–Hin enhancer interactions and from the crystal structure of FIS, in which helix-turn-helix DNA binding motifs in the C-terminal regions of FIS subunits bind DNA (Bruist et al., 1987; Kostrewa et al., 1991; Yuan et al., 1991). Since the

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FIS–RNA polymerase interaction

Figure 8. Effect of FIS sites II and III on the in vivo activities of promoters containing or lacking site I. b-Galactosidase activities were determined (see Materials and Methods) for E. coli strains containing single copy promoter–lacZ fusions illustrated on the left. The activities of the fusions were normalized to that of the rrnB P1 [ − 88 to + 50, D − 72]-lacZ fusion (A) in each strain background (1450 and 5970 Miller units in the fis + and fis − strains, respectively). Activities of rrnB P1-lacZ fusions which lack FIS binding sites are greater in a fis − than in a fis + strain as a consequence of feedback derepression of rRNA synthesis (Ross et al., 1990). Therefore, it is not valid to compare the b-galactosidase activities of fusions in fis − strains directly with identical fusions in fis + strains. The extent of activation by FIS depends somewhat on growth phase and media conditions (A. Appleman, W. Ross and R. L. Gourse, unpublished). Therefore, all lysogens were grown under identical conditions, and promoter activities were determined at the same optical density.

DNA recognition helices in the protein are too closely spaced relative to the periodicity of the major groove to permit binding to straight B-form DNA, the DNA binding site was proposed to bend 070° to accommodate them (Yuan et al., 1991; Kostrewa et al., 1991). FIS induces a substantial DNA bend at rrnB P1 FIS site I (Gosink et al., 1993) as well as at other FIS binding sites (Finkel & Johnson, 1992). The variable extent of hydroxyl radical protection observed in the flanking regions of the three FIS recogniton sequences in the rrnB P1 UAR (Figure 3) is consistent with the finding that flanking sequences wrap around FIS, and that the extent of wrapping is dependent on the sequence of the flanking regions (Pan et al., 1994). FIS-induced DNA bending may be important for the proper positioning of FIS and RNAP since a FIS mutant with altered DNA bending properties poorly activates rrnB P1 (Gosink et al., 1993). If FIS bends all three rrnB P1 FIS sites, the FIS dimers bound at the three sites could lie on the inner surface of a DNA loop. However, this simple model is complicated by the presence of a sequence-directed bend centered at approximately − 100 (Gaal et al., 1994). At present it is not clear if this intrinsic curvature is maintained when FIS is bound or if it is in phase with the FIS-induced DNA bend.

Several lines of evidence presented here and in previous reports suggest that FIS and RNAP interact directly at rrnB P1. (1) Since site I, contacted by FIS, and the UP element, contacted by RNAP, are separated by only one helical turn (Figures 4, 5), the protein surfaces may be in close proximity. (2) The presence of RNAP apparently results in better protection of the promoter-proximal end of site I by FIS (Figure 4 and data not shown). (3) Binding of FIS and RNAP is reciprocally cooperative: RNAP enhances the affinity of FIS for site I, and FIS enhances the affinity of RNAP for rrnB P1 (Figures 6, 7). Although the cooperativity displayed by the two proteins theoretically could be mediated by changes in DNA conformation rather than by direct contacts, this would require that each protein induce topological changes in the DNA, which would facilitate the binding of the other. (4) FIS-mediated activation of rrnB P1 is strongly dependent on the rotational orientation of site I with respect to the core promoter (Newlands et al., 1992; Zacharias et al., 1992). (5) A mutant FIS protein was identified which does not activate rrnB P1 in vitro under conditions where it binds and bends site I (Gosink et al., 1993), implying that another function of FIS (e.g. interaction with RNAP) is required for activation. The interacting surfaces of FIS and RNAP remain to be defined. Models of the FIS-DNA complex suggest that the N-terminal domain of FIS would be available for interaction with other proteins (Kostrewa et al., 1991; Yuan et al., 1991; Pan et al., 1994). Genetic and biochemical data indicate that residues within the N-terminal half of FIS are likely to interact with the Hin recombinase (Osuna et al., 1991). Likewise, since a mutation in the N-terminal region of FIS eliminates its ability to activate rrnB P1, but not its ability to bind and bend DNA (Gosink et al., 1993), it is possible that residues in the N-terminal portion of FIS might contact RNAP. The C-terminal domains of the a and s subunits of RNAP have been implicated as contact regions for transcription activator proteins (Ishihama, 1993; Ebright, 1993; Li et al., 1994). Since RNAP contacts the UP element through the C-terminal domain (CTD) of a (Ross et al., 1993; Blatter et al., 1994), and since the UP element and FIS site I are adjacent, it is tempting to speculate that RNAP contacts FIS through the CTD of a. However, two lines of evidence suggest that this is not the case. First, FIS activates rrnB P1 in vitro even when RNAP contains a subunits lacking the CTD (Ross et al., 1993). Second, the UP element DNA sequences are not required for FIS-mediated activation of rrnB P1 in vitro and in vivo (Rao et al., 1994). Thus, if the a subunit interacts with FIS, it would have to do so through its N-terminal domain, which has not been implicated previously as a response domain for activator proteins. Alternatively, FIS might interact with another subunit of RNAP. In the model illustrated in Figure 5, the centers of the protected surfaces in FIS site I and those in the

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Figure 9. DNase I footprint titration of site I in the presence and absence of sites II and III. Equilibrium mixtures containing either a DNA template with rrnB P1 sequences − 88 to + 50 (A) or − 154 to + 50 (B) were incubated with varying amounts of FIS for 10 min and then treated with 5 mg/ml DNase I for 30 s. Reactions in lanes 1 to 11 in A and B contained FIS at 0 nM, 0 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.6 nM, 0.8 nM, 1.2 nM, 2.4 nM or 7.2 nM, respectively. Diagnostic and reference signals used for quantitation are indicated by filled and hollow arrows, respectively. Reactions were performed in 150 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2 and 100 mg/ml BSA at 22°C. The DNA templates, [SmaI( − 138) to HindIII( + 50)] from pSL9 and [SmaI( − 204) to HindIII( + 50)] from pSL7 were 32P-labeled in the top strand at + 50. C, Binding isotherms derived from the titration experiments shown in A (circles; − sites II and III) and B (triangles; + sites II and III).

UP element are offset by a rotation of about 90°. In addition, the FIS-induced DNA bend and deviations in DNA structure from typical B-form are not considered in the model and might further affect the relative orientation of the DNA surfaces binding the C-terminal regions of a and FIS. Thus, the interacting surface of FIS might in fact be closer to another part of RNAP rather than the CTD of a, explaining the absence of a requirement for the UP element and the aCTD in activation by FIS. Transcription from rrnB P1 in vitro is very sensitive to the salt concentration (Gourse, 1988), and the effect of FIS on transcription decreases as the cation concentration is decreased (Ross et al., 1990). The titration experiments were performed under conditions (linear DNA template, 120 mM NaCl, 5 nM RNAP) in which the effect of FIS on the binding constant for RNAP (Figure 7) is roughly comparable to the observed effect of FIS on transcription under the same conditions (A. J. Bokal, unpublished results). The effects of FIS on the initial binding equilibrium may be sufficient to explain the effects of FIS site I on transcription in vivo. Nevertheless, although our experiments indicate that FIS facilitates the initial binding of RNAP at rrnB P1, our findings do not rule out the possibility that FIS could also affect subsequent steps in the initiation mechanism under some conditions in vitro or in vivo, as suggested by others (Sander et al., 1993).

Role of sites II and III in activation We found that FIS dimers bound at the three sites in rrnB P1 do not interact cooperatively. Similarly, cooperative interactions were not observed between FIS dimers at the two FIS sites in the Hin recombinational enhancer (Johnson et al., 1987). FIS dimers bound at rrnB P1 FIS sites II and III appear to stimulate RNAP directly, and we assume that their mechanism of activation in the absence of FIS bound at site I is the same as that observed when site I is present. Thus, if FIS dimers bound at each of the three FIS sites interact simultaneously with RNAP, the interactions might involve different surfaces on RNAP. It remains to be seen whether the distal FIS sites might have a regulatory role in addition to their relatively small effect on rRNA transcription during exponential growth. However, in vivo footprinting analyses indicate that although occupancy of the UAR by FIS varies in different stages of growth, the filling of sites I, II, and III varies together (W. Ross, J. Salomon & R. L. Gourse, unpublished data).

Materials and Methods Bacterial strains and plasmids Strains monolysogenic for l carrying rrnB P1-lacZ fusions were described previously or were constructed as described (Gourse et al., 1986; Gaal et al., 1989). RLG733

205

Mechanism of Activation of rRNA Transcription by FIS

(Ross et al., 1990), RLG2265 (Rao et al., 1994), RLG1760 and RLG1758 are l lysogens of NK5031 (DlacM5262 supF Nal R ) which carry fusions of rrnB P1 [ − 88 to + 50], rrnB P1 [ − 88 to + 50, D − 72], rrnB P1 [ − 154 to + 50], and rrnB P1 [ − 154 to + 50, D − 72] to lacZ, respectively. (The numbers in brackets refer to the limits of rrnB P1 sequence with respect to the transcription start site.) The D − 72 derivatives of rrnB P1 contain a single bp deletion at postion − 72 and were constructed by site-directed mutagenesis (Kunkel, 1985). Derivatives of these strains containing fis::kan 767 (Johnson et al., 1988) were constructed by P1 transduction (Ross et al., 1986). RLG1779, RLG1780, RLG1766 and RLG1762 are fis::kan 767 derivatives of RLG733, RLG2265, RLG1760 and RLG1758, respectively. The plasmids pSL9 and pSL7 (Rao et al., 1994) contain rrnB P1 [ − 88 to + 50] and rrnB P1 [ − 154 to + 50], respectively, inserted into the multiple cloning site of pSL6 (Gosink et al., 1993). The pJNBend130 plasmid, a pUC19 derivative, contains rrnB P1 [ − 154 to − 28] as an EcoRI-BamHI insert in the multiple cloning site (Gaal et al., 1994). General methods DNA fragments used for footprinting were derived from pSL9, pSL7 or pJNBend130. Plasmids were purified using Qiagen columns (Qiagen, Inc.), phenol extracted, digested with a restriction enzyme, and 3' end-labeled using phage T7 RNAP (Sequenase, USB) and an [a-32P]dNTP. Single-end labeled fragments were produced by digestion with a second restriction enzyme, isolated on polyacrylamide gels, eluted from gel slices by diffusion, and purified over benzoylated-napthalated DEAE-cellulose (Boehringer-Mannheim). Purified FIS and RNAP were provided by Reid Johnson (UCLA) and by Dayle Hager and Richard Burgess (UW-Madison), respectively. RNAP was approximately 40% active (Leirmo & Gourse, 1991). When necessary, proteins were diluted in 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, and 30% (v/v) glycerol. FIS and RNAP concentrations are expressed in terms of monomeric protein and active protein, respectively. Footprinting Protein–DNA complexes were prepared in 25 ml reactions as described in the legends for Figures 2, 4, 6, 7, and 9. To ensure uniformity among reactions in each experiment, the end-labeled DNA fragment and buffer components of each reaction were distributed from a single mixture. Hydroxyl radical (Newlands et al., 1991), dimethyl sulfate (Newlands et al., 1991), and DNase I (Ross et al., 1990) treatment, processing, and electrophoresis were performed as described previously. Sequence markers were prepared as described (Ross et al., 1990). For DNase I footprint titrations, densitometry of autoradiograms was performed with a Hoefer GS300 linear densitometer and GS365 software. Exposures were analyzed with signals in the linear film response range. Fractional site saturation (f) was quantified as: f=1−

Y Y0

(1)

where Y is the sum of the intensities of a group of signals within the binding site of the titrating protein and Y0 is the intensity of these signals in the absence of the protein. To control for recovery of cleaved fragments, Y was calculated

by normalizing the intensity of signals within the binding site of the titrating protein (diagnostic signals) to the intensity of signals outside the binding site (reference signals). Since each point on a binding isotherm, a plot of f versus protein concentration, depends on the value of Y0 , we obtained accurate estimates of Y0 by fitting Y versus P to:

$

Y = Y0 × 1 −

KD × P 1 + KD × P

%

(2)

where Y and Y0 are as defined above (1), KD is the equilibrium dissociation constant and P is the concentration of the titrating protein. Estimates of equilibrium dissociation constants were obtained by fitting f versus P to: f=

KD × P 1 + KD × P

(3)

where f, KD and P are as defined above (1, 2). Fitting to equations (2) and (3), derived from the Langmuir isotherm, is justified since less than 10% of the titrating protein is site-bound in the reactions (Brenowitz et al., 1986). Curves were fit with SigmaPlot v4.0 software (Jandel Scientific). Replicate titration experiments yielded nearly identical binding isotherms and estimates of KD with standard deviations of 010%. Representative experiments are presented in Results. b-Galactosidase determinations Determinations of b-galactosidase synthesis directed by rrnB P1-lacZ transcriptional fusions were made as described (Miller, 1972). Lysogens were grown logarithmically for at least three generations in LB at 30°C and harvested at an A600 of 00.3. Duplicate measurements were made for each of two independent cultures of each lysogen.

Acknowledgements We thank Julia Salomon for excellent technical assistance and Paula Schlax, Tom Record, and Gary Ackers for helpful comments. This work was supported by RO1 GM37048 (R.L.G.) and by a Cell and Molecular Biology predoctoral training grant (A.J.B.) from the National Institutes of Health.

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Edited by R. Schleif (Received 5 July 1994; accepted 3 October 1994)