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. 2002 Oct 1;16(19):2557–2565. doi: 10.1101/gad.237502

Requirement for two copies of RNA polymerase α subunit C-terminal domain for synergistic transcription activation at complex bacterial promoters

Georgina S Lloyd 1, Wei Niu 2,3, John Tebbutt 1, Richard H Ebright 2, Stephen JW Busby 1,4
PMCID: PMC187446  PMID: 12368266

Abstract

Transcription activation by the Escherichia coli cyclic AMP receptor protein (CRP) at different promoters has been studied using RNA polymerase holoenzyme derivatives containing two full-length α subunits, or containing one full-length α subunit and one truncated α subunit lacking the α C-terminal domain (αCTD). At a promoter having a single DNA site for CRP, activation requires only one full-length α subunit. Likewise, at a promoter having a single DNA site for CRP and one adjacent UP-element subsite (high-affinity DNA site for αCTD), activation requires only one full-length α subunit. In contrast, at promoters having two DNA sites for CRP, or one DNA site for CRP and two UP-element subsites, activation requires two full-length α subunits. We conclude that a single copy of αCTD is sufficient to interact with one CRP molecule and one adjacent UP-element subsite, but two copies of αCTD are required to interact with two CRP molecules or with one CRP molecule and two UP-element subsites.

Keywords: Transcription activation, cAMP receptor protein, RNA polymerase α subunit, CRP-dependent promoters

Escherichia coli RNA polymerase holoenzyme (RNAP) has subunit composition α2ββ‘ως (Ebright 2000). The major determinants of RNAP for promoter recognition reside in the ς subunit, which makes direct sequence-specific contacts with promoter −10 and −35 elements (for review, see Busby and Ebright 1994; Gross et al. 1998). However, at many promoters, the RNAP α subunit also plays an important role in promoter recognition (for review, see Busby and Ebright 1994; Gourse et al. 2000). The RNAP α subunit consists of two separate domains connected by a flexible linker (Blatter et al. 1994; Jeon et al. 1995). The main function of the RNAP α subunit N-terminal domain (αNTD; residues 8–235) is to provide a scaffold for the assembly of RNAP, whereas the main function of the RNAP α subunit C-terminal domain (αCTD; residues 249–329) is to interact with promoter DNA to increase the initial binding of RNAP. At many promoters, αCTD interacts with one or more ∼9-bp A/T-rich DNA sequences located upstream of the −35 element (UP-element subsites; Estrem et al. 1999; Gourse et al. 2000). Each of the two copies of αCTD in RNAP—αCTDI and αCTDII—can interact independently with a single UP-element subsite (Estrem et al. 1999), with residues 264–269 and 296–302 (265 determinant; Gaal et al. 1996; Murakami et al. 1996) making direct contact with the DNA minor groove (Naryshkin et al. 2000; Ross et al. 2001; Yasuno et al. 2001).

A second function of αCTD is to serve as a target for transcriptional activators. One such transcriptional activator is the E. coli cAMP receptor protein [CRP; also referred to as catabolite activator protein (CAP)], which activates >100 genes in response to glucose starvation and other stresses (for review, see Kolb et al. 1993). The activity of CRP is triggered by binding of cAMP. CRP functions as a homodimer and, at target promoters, binds and sharply bends a 22-bp twofold-symmetric DNA site (Schultz et al. 1991; Parkinson et al. 1996). CRP activates transcription initiation at most target promoters by making direct protein–protein interactions with αCTD that recruits αCTD, and hence the rest of RNAP, to promoter DNA (for review, see Busby and Ebright 1994, 1999). Mutational analysis indicates that the determinant of CRP responsible for CRP–αCTD interaction is an ∼14 × 11 Å surface located adjacent to the helix-turn-helix DNA-binding motif of CRP [activating region 1 (AR1); residues 156–164; Niu et al. 1994]. Single amino-acid substitutions in AR1 (e.g., TA158, HL159) interfere with CRP–αCTD interaction and reduce the ability of CRP to activate transcription, although they do not affect the ability of CRP to bind cAMP, to bind to DNA, or to bend DNA. Mutational analysis of αCTD indicates that the determinant of αCTD responsible for CRP–αCTD interaction is an ∼20 × 10 Å surface located adjacent to the DNA-binding motif of αCTD (287 determinant; residues 285–290 and 315–318; Savery et al. 1998, 2002). Single amino-acid substitutions in the 287 determinant (e.g., VA287, EA288) interfere with the CRP–αCTD interaction and reduce CRP-dependent transcription, although they do not affect activator-independent transcription.

Simple CRP-dependent promoters (i.e., promoters that have only one DNA site for CRP) can be grouped into two classes based on the position of the DNA sites for CRP (Busby and Ebright 1999). At class I CRP-dependent promoters, the DNA site for CRP is located upstream of the core promoter (i.e., centered near positions −61, −71, −81 or −92), and CRP recruits αCTD to the DNA segment immediately downstream of the DNA site for CRP (Fig. 1A). At class II CRP-dependent promoters, the DNA site for CRP overlaps the core promoter (i.e., centered near position −41), and CRP recruits αCTD to the DNA segment immediately upstream of the DNA site for CRP (and also interacts with αNTD; Fig. 1B; Niu et al. 1996). At each of these classes of CRP-dependent promoters, CRP interacts with only one of the two copies of αCTD in RNAP (Zou et al. 1993; Busby and Ebright 1999; Niu 1999; W. Niu and R.H. Ebright, unpubl.). Therefore, in principle, the second copy of αCTD is available for potential interactions with a second molecule of CRP, or a molecule of another activator that functions by interacting with αCTD (Belyaeva et al. 1998; Busby and Ebright 1999; Langdon and Hochschild 1999).

Figure 1.

Figure 1

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Transcription activation at class I and class II cyclic AMP receptor protein (CRP)-dependent promoters: published models. (A) Ternary complex of CRP, Escherichia coli RNA polymerase holoenzyme (RNAP), and a class I CRP-dependent promoter [e.g., CC(-61.5); adapted from Blatter et al. 1994; Zhou et al. 1994a,b; Busby and Ebright 1999]. Transcription activation involves direct protein–protein interaction (black filled circle) between AR1 of the downstream subunit of CRP and the 287 determinant of α C-terminal domain (αCTD). (B) Ternary complex of CRP, RNAP, and a class II CRP-dependent promoter [e.g., CC(-41.5); adapted from Niu et al. 1996; Busby and Ebright 1999]. Transcription activation involves both (1) direct protein–protein interaction (black filled circle) between AR1 of the upstream subunit of CRP and the 287 determinant of αCTD, and (2) direct protein–protein interaction (grey filled circle) between AR2 in the downstream subunit of CRP and α subunit N-terminal domain (αNTD).

The major aim of the work presented here has been to obtain direct evidence that although only one copy of αCTD is required for CRP-dependent transcription at promoters having one DNA site for CRP, two copies of αCTD are required for CRP-dependent transcription at promoters having two DNA sites for CRP. To achieve this aim, we analyzed CRP-dependent transcription at a class I CRP-dependent promoter [CC(-61.5)], at a class II CRP-dependent promoter [CC(-41.5)], and at derivatives thereof having a second DNA site for CRP, using RNAP containing two full-length α subunits or containing one full-length α subunit and one truncated α subunit lacking αCTD.

A second aim of this work has been to address a long-standing point of contention, namely, whether a single copy of αCTD is able to interact productively with both CRP and DNA. We have proposed that αCTD contacts CRP and DNA through distinct nonoverlapping determinants (with the 287 determinant contacting CRP, and the 265 determinant contacting DNA; Savery et al. 1998, 2002; Busby and Ebright 1999), and that αCTD productively contacts both CRP and DNA in CRP-dependent transcription complexes (Blatter et al. 1994; Busby and Ebright 1994, 1999; Tang et al. 1994; Zhou et al. 1994b; Belyaeva et al. 1998; Savery et al. 1998, 2002). However, Ishihama and others, following a study of substitutions in αCTD that reduce CRP-dependent activation of the lac promoter, proposed that αCTD contacts CRP and DNA through a single determinant (or two extensively overlapping determinants). Thus, one copy of αCTD can contact CRP or DNA but cannot contact both CRP and DNA (Murakami et al. 1996; Ishihama 1997; Ozoline et al. 2000). To resolve this issue, we analyzed CRP-dependent transcription by an RNAP derivative having only one full-length α subunit at a class II CRP-dependent promoter with a single UP-element subsite located immediately adjacent to the DNA site for CRP.

Results

Full activation at a CRP-dependent promoter having two DNA sites for CRP requires both copies of αCTD: class I promoter derivative having a second DNA site for CRP

To determine the number of copies of αCTD required for CRP-dependent transcription at a class I promoter derivative having a second DNA site for CRP, we performed experiments with the CC(-61.5) promoter, a class I CRP-dependent promoter having a consensus DNA site for CRP centered at position −61.5 (Fig. 2A, top; Gaston et al. 1990), and with the CC(-93.5)CC(-61.5) promoter, a derivative of CC(-61.5) having an additional consensus DNA site for CRP centered at position −93.5 (Fig. 2A, bottom; Tebbutt et al. 2002). The presence of the additional DNA site for CRP in CC(-93.5)CC(-61.5) results in a higher level of CRP-dependent transcription in vitro (Fig. 3, lanes 2,8) and in vivo (three- to fourfold synergistic effect; Table 1; Joung et al. 1993; Langdon and Hochschild 1999; Tebbutt et al. 2002).

Figure 2.

Figure 2

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Promoters analyzed in this work. Promoter −10 and −35 elements are shown as open boxes; DNA sites for cyclic AMP receptor protein (CRP), as shaded boxes (with critical TGTGA/TCACA elements in boldface), and 6-bp A/T-tracts constituting UP-element subsites are doubly underlined and in boldface. (A) Class I CRP-dependent promoter and derivative with second DNA site for CRP. (B) Class II CRP-dependent promoter and derivative with second DNA site for CRP. (C) CRP-dependent promoter derivative with one UP-element subsite adjacent to DNA site for CRP. (D) CRP-dependent promoter derivative with two UP-element subsites—one adjacent to DNA site for CRP, and one not adjacent to DNA site for CRP.

Figure 3.

Figure 3

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Full activation at a cyclic AMP receptor protein (CRP)-dependent promoter having two DNA sites for CRP requires both copies of α C-terminal domain (αCTD): Class I promoter derivative having a second DNA site for CRP. The figure shows results of transcription experiments at CC(-61.5) (lanes 1–6; sequence in Fig. 2A, top) and CC(-93.5)CC(-61.5) (lanes 7–12; sequence in Fig. 2A, bottom), using Escherichia coli RNA polymerase holoenzyme (RNAP) derivatives with two full-length α subunits (αI/αII) and with one full-length and one truncated α subunit (αI/αΔII). Yields of transcripts from CC(-61.5) and CC(-93.5)CC(-41.5) are normalized with reference to the control RNA I transcript.

Table 1.

Activity of different promoters

Promoter
Expression (β-galactosidase units)
CC(-61.5) 390
CC(-93.5)CC(-61.5) 1510
CC(-41.5) 460
CC(-90.5)CC(-41.5) 1600
X(-59.5)CC(-41.5) 410
α(-59.5)CC(-41.5) 2585
CC(-69.5)α(-51.5)α(-41.5) 1450
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Table 1 shows β-galactosidase activities in DH5α Δlac cells carrying the lac expression vector plasmid, pRW50, containing different promoters, as listed. The measured activities (listed in standard Miller units) give an estimation of in vivo cyclic AMP receptor protein (CRP)-dependent promoter activity (Lodge et al. 1992). Activities were measured in cells grown aerobically in L-broth containing 35 μg/mL tetracycline exactly as in our previous studies (Law et al. 1999; Tebbutt et al. 2002) 

We performed experiments using RNAP derivatives having two full-length α subunits, or one full-length α subunit and one truncated α subunit lacking αCTD. We prepared RNAP derivatives by co-expressing, respectively, genes encoding α, β, β‘, ω, ς70, and hexahistidine-tagged α, or genes encoding α, β, β‘, ω, ς70, and hexahistidine-tagged [RA45]α(1–235) (wherein the RA45 substitution prevents an α derivative from occupying the β-associated αI site within RNAP, and thereby restricts an α derivative to the β‘-associated αII site within RNAP; Murakami et al. 1997; Estrem et al. 1999; Niu 1999), lysing cells, and performing metal ion-affinity chromatography (Niu et al. 1996; Estrem et al. 1999; Niu 1999).

Figure 3, lanes 1–6, present results of transcription experiments with CC(-61.5) and RNAP derivatives having two full-length α subunits, or one full-length α subunit and one truncated α subunit lacking αCTD. The results verify that transcription at CC(-61.5) is CRP-dependent and AR1-dependent (Fig. 3, lanes 1–3). In addition, the results show that transcription at CC(-61.5) is unaffected by truncation of one α subunit (Fig. 3, lanes 2,5), indicating that only one copy of αCTD is required at CC(-61.5).

Figure 3, lanes 7–12, presents results of parallel transcription experiments with CC(-93.5)CC(-61.5). At CC(-93.5)CC(-61.5), as at CC(-61.5), transcription is CRP-dependent and AR1-dependent (Fig. 3, lanes 7–9). However, in contrast to the situation at CC(-61.5), transcription at CC(-93.5)CC(-61.5) is reduced by truncation of one α subunit (Fig. 3, lanes 8,11)—with the synergistic effect of the second DNA site for CRP being, within error, lost (Fig. 3, lanes 5,11)—indicating that two copies of αCTD are required for full, synergistic activation at CC(-93.5)CC(-61.5).

Full activation at a CRP-dependent promoter having two DNA sites for CRP requires both copies of αCTD: class II promoter derivative having a second DNA site for CRP

To determine the number of copies of αCTD required for CRP-dependent transcription at a class II promoter derivative having a second DNA site for CRP, we performed experiments comparing the CC(-41.5) promoter, a class II CRP-dependent promoter having a consensus DNA site for CRP centered at position −41.5 (Gaston et al. 1990; Fig. 2B, top), and the CC(-90.5)CC(-41.5) promoter, a derivative of CC(-41.5) having an additional consensus DNA site for CRP centered at position −90.5 (Fig. 2B, bottom; Busby et al. 1994; Belyaeva et al. 1998). The presence of the additional DNA site for CRP in CC(-90.5)CC(-41.5) results in a higher level of CRP-dependent transcription in vitro (Fig. 4, lanes 2,8) and in vivo (three- to fourfold synergistic effect; Table 1; Busby et al. 1994; Belyaeva et al. 1998).

Figure 4.

Figure 4

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Full activation at a cyclic AMP receptor protein (CRP)-dependent promoter having two DNA sites for CRP requires both copies of α C-terminal domain (αCTD): Class II promoter derivative having a second DNA site for CRP. Results of transcription experiments at CC(-41.5) (lanes 1–6; sequence in Fig. 2B, top) and CC(-90.5)CC(-41.5) (lanes 7–12; sequence in Fig. 2B, bottom), using Escherichia coli RNA polymerase holoenzyme (RNAP) derivatives with two full-length α subunits (αI/αII) and with one full-length and one truncated α subunit (αI/αΔII). Yields of transcripts from CC(-41.5) and CC(-90.5) CC(-41.5) are normalized with reference to the control RNA I transcript.

Figure 4, lanes 1–6, presents results of transcription experiments with CC(-41.5) and RNAP derivatives having two full-length α subunits, or one full-length α-subunit and one truncated α-subunit lacking αCTD. The results verify that transcription at CC(-41.5) is CRP-dependent and AR1-dependent (Fig. 4, lanes 1–3). In addition, the results show that transcription at CC(-41.5) is unaffected by truncation of one α subunit (Fig. 4, lanes 2,5), indicating that only one copy of αCTD is required at CC(-41.5).

Figure 4, lanes 7–12, present results of parallel transcription experiments with CC(-90.5)CC(-41.5). At CC(-90.5)CC(-41.5), transcription is CRP-dependent and AR1-dependent (Fig. 4, lanes 7–9), as at CC(-41.5). However, in contrast to the situation at CC(-41.5), transcription at CC(-90.5)CC(-41.5) is reduced by truncation of one α subunit (Fig. 4, lanes 8,11)—with the synergistic effect of the second DNA site for CRP being, within error, fully lost (Fig. 4, lanes 5,11)—indicating that two copies of αCTD are required for full synergistic activation at CC(-90.5)CC(-41.5).

Full activation at a CRP-dependent promoter having a DNA site for CRP and one adjacent UP-element subsite requires only one copy of αCTD

To determine the number of copies of αCTD required for CRP-dependent transcription at a promoter having a single DNA site for CRP and one adjacent UP-element subsite, we performed experiments with the α(-59.5)CC(-41.5) promoter (Fig. 2C), a derivative of CC(-41.5) having a consensus UP-element subsite (Estrem et al. 1999) positioned adjacent to the DNA site for CRP and phased optimally relative to the DNA site for CRP (T6:A6 tract centered 18 bp from the centre of the DNA site for CRP; see Lloyd et al. 1998). The presence of the consensus UP-element subsite in α(-59.5)CC(-41.5) results in a greater than fivefold higher level of CRP-dependent transcription relative to X(-59.5)CC(-41.5), a derivative of CC(-41.5) having a DNA sequence with no specific determinants for interaction with αCTD positioned adjacent to the DNA site for CRP [“No-UP” sequence of Estrem et al. (1999); greater than fivefold “synergistic effect”; Table 1; Lloyd et al. 1998].

Figure 5 presents results of transcription experiments with α(-59.5)CC(-41.5) and RNAP derivatives having two full-length α subunits, or one full-length α subunit and one truncated α subunit lacking αCTD. The results verify that transcription at α(-59.5)CC(-41.5) is CRP-dependent and AR1-dependent (Fig. 5, lanes 1–3). In addition, the results show that transcription at α(-59.5)CC(-41.5) is unaffected by truncation of one α subunit (Fig. 5, lanes 2,5), indicating that only one copy of αCTD is required for full synergistic activation at α(-59.5)CC(-41.5). We conclude that a single copy of αCTD is sufficient for functional interaction with both CRP and an adjacent UP-element subsite.

Figure 5.

Figure 5

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Full activation at a cyclic AMP receptor protein (CRP)-dependent promoter having a DNA site for CRP and one adjacent UP-element subsite requires only one copy of α C-terminal domain (αCTD). Results of transcription experiments at α(-59.5)CC(-41.5) (sequence in Fig. 2C) using Escherichia coli RNA polymerase holoenzyme (RNAP) derivatives with two full-length α subunits (αI/αII) and with one full-length and one truncated α subunit (αI/αΔII). Yields of transcripts from α(-59.5)CC(-41.5) are normalized with reference to the control RNA I transcript.

Full activation at a CRP-dependent promoter having a DNA site for CRP and two UP-element subsites requires two copies of αCTD

To determine the number of copies of αCTD required for CRP-dependent transcription at a promoter having a single DNA site for CRP and two UP-element subsites, we performed experiments with the CC(-69.5)α(-51.5)α(-41.5) promoter (Fig. 2D), a promoter having a DNA site for CRP centered at position −69.5 and the rrnB P1 UP-element, which contains two UP-element subsites (Estrem et al. 1999), positioned immediately downstream of the DNA site for CRP and phased optimally relative to the DNA site for CRP (UP-element subsites centered 18 and 28 bp from the center of the DNA site for CRP; Lloyd et al. 1998). The presence of the rrnB P1 UP-element results in a greater than fivefold higher level of CRP-dependent transcription (Table 1; Savery et al. 1995; Law et al. 1999).

Figure 6 presents results of transcription experiments with CC(-69.5)α(-51.5)α(-41.5) and RNAP derivatives having two full-length α subunits, or one full-length α subunit and one truncated α subunit lacking αCTD. The results verify that transcription at CC(-69.5)α(-51.5)α(-41.5) is CRP-dependent and AR1-dependent (Fig. 6, lanes 1–3). In addition, the results show that transcription at CC(-69.5)α(-51.5)α(-41.5) is reduced by truncation of one α subunit (Fig. 6, lanes 2,5), indicating that two copies of αCTD are required for full, synergistic activation at CC(-69.5)α(-51.5)α(-41.5). Surprisingly, with the RNAP derivatives having just one full-length α subunit, levels of CRP-dependent transcription are very low. This implies that transcription activation at CC(-69.5)α(-51.5)α(-41.5) requires the binding of αCTD both at position −41.5 and at position −51.5 (Fig. 7D).

Figure 6.

Figure 6

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Full activation at a cyclic AMP receptor protein (CRP)-dependent promoter having a DNA site for CRP and two UP-element subsites requires two copies of α C-terminal domain (αCTD). Results of transcription experiments at CC(-69.5)α(-51.5)α(-41.5) (sequence in Fig. 2D) using Escherichia coli RNA polymerase holoenzyme (RNAP) derivatives with two full-length α subunits (αI/αII) and with one full-length and one truncated α subunit (αI/αΔII). Yields of transcripts from CC(-69.5)α(-51.5)α(-41.5) are normalized with reference to the control RNA I transcript.

Figure 7.

Figure 7

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Models. α C-terminal domain (αCTD), α subunit N-terminal domain (αNTD), β, β’, ω, and ς denote, respectively, the Escherichia coli RNA polymerase holoenzyme (RNAP) α subunit C-terminal domain, the RNAP α subunit N-terminal domain, and the RNAP β, β‘, ω, and ς70 subunits. Positions of centres of DNA sites for cyclic AMP receptor protein (CRP), UP-element subsites, promoter −35 elements, promoter −10 elements, and transcription start sites are numbered. Functional AR1–αCTD, AR2–αNTD, and (UP-element subsite)–αCTD interactions are indicated by, respectively, black filled circles, grey filled circles, and black filled bars. For clarity, copies of αCTD not involved in interactions with AR1 of CRP or an UP-element subsite are drawn in a raised position, and DNA is drawn as a straight line. [In fact, copies of αCTD not involved in AR1–αCTD or (UP-element subsite)–αCTD interactions are likely to make nonspecific DNA–αCTD interactions with upstream DNA (Busby and Ebright 1999; Naryshkin et al. 2001), and both CRP and RNAP are known to bend DNA (Schultz et al. 1991; Rees et al. 1993; Parkinson et al. 1996)]. (A) Transcription activation at a class I CRP-dependent promoter (one copy of αCTD required) and at a derivative having a second DNA site for CRP (two copies of αCTD required). (B) Transcription activation at a class II CRP-dependent promoter (one copy of αCTD required) and at a derivative having a second DNA site for CRP (two copies of αCTD required). (C) Transcription activation at a CRP-dependent promoter having one UP-element subsite adjacent to DNA site for CRP (one copy of αCTD required). (D) Transcription activation at a CRP-dependent promoter having two UP-element subsites—one adjacent to DNA site for CRP, and one not adjacent to DNA site for CRP (two copies of αCTD required).

Discussion

Our results establish that although only one copy of αCTD is required for full activation at a CRP-dependent promoter having only one DNA site for CRP (in accord with Zou et al. 1993; Zhou et al. 1994b; Niu 1999), two copies of αCTD are required for full activation at a CRP-dependent promoter having two DNA sites for CRP (Figs. 3, 4). Furthermore, our results establish that although only one copy of αCTD is required for full activation at a CRP-dependent promoter with a DNA site for CRP and an adjacent single UP-element subsite (Fig. 5), two copies of αCTD are required for full activation at a CRP-dependent promoter with a DNA site for CRP and two UP-element subsites (Fig. 6). Our conclusions are based on experiments with preparations of RNAP in which the β-associated α subunit, αI, was full-length, and the β‘-associated α subunit, αII, was truncated. Extensive data establish that αCTD of αI and αCTD of αII can function interchangeably—at both class I and class II CRP-dependent promoters (Niu 1999; W. Niu and R.H. Ebright, unpubl.; cf. Estrem et al. 1999). Therefore, we are confident that identical results would have been obtained in experiments analyzing an RNAP derivative in which the β-associated α subunit, αI, was truncated, and the β‘-associated α subunit, αII, was full-length.

Our results document a strikingly simple mechanism for synergistic effects of two activator molecules, or of an activator molecule and a nonadjacent UP-element subsite, namely, simultaneous interactions with the two copies of αCTD in RNAP (Fig. 7).

Our finding that a single αCTD suffices to manifest the full synergistic effect of CRP and an adjacent single UP-element subsite (Fig. 5, lanes 2,5) has a further implication; namely, a single αCTD can interact productively with both CRP and DNA. This supports the proposal that αCTD has distinct, nonoverlapping functional determinants for interaction with CRP and DNA, and that αCTD interacts simultaneously with CRP and DNA in CRP-dependent transcription complexes (Blatter et al. 1994; Busby and Ebright 1994, 1999; Tang et al. 1994; Zhou et al. 1994b; Savery et al. 1998, 2002) and is less easy to reconcile with the alternative proposal that the same surface of αCTD interacts with both CRP and DNA (Murakami et al. 1996; Ishihama 1997; Ozoline et al. 2000). We suggest that αCTD interacts simultaneously with activator and DNA in many, possibly all, activator-dependent, αCTD-dependent transcription complexes.

Materials and methods

Strains, plasmids, and promoter derivatives

Bacterial strains, plasmids, and promoter derivatives used in this study are listed in Table 2. EcoRI-HindIII fragments carrying promoter derivatives were cloned in vector plasmid pSR.

Table 2.

Bacterial strains, plasmids and promoter fragments

Name
Brief description
Source/Reference
Strains
 DH5α E. coli Δlac recA− Jessee 1986
 XL1-blue E. coli recAI [F′ lac1q] Bullock et al. 1987
Plasmids
 pSR pBR322 derivative containing transcription terminator Kolb et al. 1995
 pREII-NHα Plasmid encoding N-terminally hexahistidine-tagged α under control of the tandem lppP-lacPUV5 promoters Niu et al. 1996
 pREII-NHα45A[1-235] Plasmid encoding N-terminally hexahistidine-tagged [Ala-45]α(1-235) under control of the tandem lppP-lacPUV5 promoters Niu 1999
 pRW50 Low copy number, broad host range lac expression vector carrying resistance to tetracycline Lodge et al. 1992
Promoters
 CC(-61.5) CRP-dependent promoter with consensus DNA site for CRP centred at position -61.5 (Fig. 2A, top) Gaston et al. 1990
 CC(-93.5)CC(-61.5) CRP-dependent promoter with consensus DNA sites for CRP centred at positions -93.5 and -61.5 (Fig. 2A, bottom) Tebbutt et al. 2002
 CC(-41.5) CRP-dependent promoter with consensus DNA site for CRP centred at position -41.5 (Fig. 2B, top) Gaston et al. 1990
 CC(-90.5)CC(-41.5) CRP-dependent promoter with consensus DNA sites for CRP centred at positions -90.5 and -41.5 [Fig. 2B, bottom; designated MI (-90.5) in Belyaeva et al. (1998)] Belyaeva et al. 1998
 α(-59.5)CC(-41.5) CRP-dependent promoter with consensus DNA site for CRP centred at position -41.5 and consensus UP-element subsite immediately upstream of the DNA site for CRP (Fig. 2C) This work
 X(-59.5)CC(-41.5) CRP-dependent promoter with consensus DNA site for CRP centred at position -41.5 and no-UP sequence (Estrem et al. 1999) immediately upstream of the DNA site for CRP This work
 CC(-69.5)α(-51.5)α(-41.5) CRP-dependent promoter with consensus DNA site for CRP centred at position -69.5 and the rrnB P1 UP-element, which contains two UP-element subsites (Estrem et al. 1999), immediately downstream of the DNA site for CRP [Fig. 2D; designated CC(-69.5)α(-44) in Law et al.(1999)] Law et al. 1999
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E. coli indicates Escherichia coli; CRP indicates cyclic AMP receptor protein. 

To measure promoter activities, the fragments were cloned into the lac expression vector, pRW50 (Lodge et al. 1992), and β-galactosidase expression in DH5α cells, carrying the different plasmids, was measured exactly as in our previous studies (Law et al. 1999; Tebbutt et al. 2002).

X(-59.5)CC(-41.5) was constructed by PCR, using the primer 5′-TTCAGATCTGACTGCAGTGGTACCTAGGAATTAAAT GTGATGTACATCACATGG-3′ to introduce the no-UP sequence of Estrem et al. (1999) upstream of the DNA site for CRP of CC(-41.5). α(-59.5)CC(-41.5) was constructed by PCR, using the primer 5′-TTCAGATCTGACTGCAGTGGTATTTTTTGT ATAAATGTGATGTACATCACATGG-3′, to insert a consensus UP-element subsite (underlined; Estrem et al. 1999) upstream of the DNA site for CRP of CC(-41.5).

RNAP derivatives

RNAP derivatives carrying two full-length α subunits (RNAP αI/αII) and carrying one full-length α subunit and one truncated α subunit lacking αCTD (RNAP αI/αΔII) were prepared from transformants of E. coli strain XL1-blue with, respectively, pREII-NHα and pREII-NHα45A(1–235), using Ni2+-NTA agarose chromatography and Mono-Q chromatography (Niu et al. 1996; Estrem et al. 1999; Niu 1999).

CRP derivatives

Wild-type CRP and CRP HL159 were prepared as in Ghosaini et al. (1988).

Transcription experiments

To measure CRP-dependent transcription activation in vitro, DNA fragments carrying promoters were cloned upstream of the bacteriophage λ oop terminator in vector plasmid pSR. Thus, CRP-dependent transcription initiation in vitro could be measured by the appearance of a transcript running from the cloned promoter to the oop terminator. Quantification was facilitated by the simultaneous appearance of the vector-encoded RNA I transcript, which was controlled by a factor-independent promoter unaffected by truncation of αCTD (Meng et al. 2000). Transcription experiments were performed in 25 μL samples containing: 0.2 nM supercoiled template DNA, 60 nM RNAP derivative, 20 nM CRP derivative (40 nM in experiments in Figs. 3, 5), 0.2 mM cAMP, 10 μM UTP, 2.5 μCi α-32[P]-UTP (3000 Ci/mmole), 200 μM ATP, 200 μM CTP, 200 μM GTP, 40 mM Tris-acetate (pH 7.9), 200 mM KCl (100 mM in experiments in Fig. 3), 10 mM MgCl2, 1 mM dithiothreitol (DTT), and 100 μg/mL bovine serum albumin. Reactions were initiated by the addition of the RNAP derivative and were terminated after 15 min at 30°C by the addition of 25 μL stop solution (7 M urea, 1% SDS, 10 mM EDTA, and 0.05% bromophenol blue, and 0.05% xylene cyanol). Products were isolated using 6% acrylamide gels containing 7 M urea and quantified by PhophorImager analysis (Molecular Dynamics Inc.; mean ± SD of at least three independent determinations).

Acknowledgments

This work was supported by a project grant from the United Kingdom Biotechnology and Biological Sciences Research Council to S.J.W.B., and by National Institutes of Health grant GM41376 and a Howard Hughes Medical Institute Investigatorship to R.H.E.

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.

Footnotes

E-MAIL s.j.w.busby@bham.ac.uk; FAX 44-121-414-7366

Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.237502.

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