Introduction

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Escherichia coli RNA polymerase holoenzyme (RNAP) has subunit composition ₂` (Ebright 2000). The major determinants of RNAP for promoter recognition reside in the  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  subunit also plays an important role in promoter recognition (for review, see Busby and Ebright 1994; Gourse et al. 2000). The RNAP  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  subunit N-terminal domain (NTD; residues 8-235) is to provide a scaffold for the assembly of RNAP, whereas the main function of the RNAP  subunit C-terminal domain (CTD; residues 249-329) is to interact with promoter DNA to increase the initial binding of RNAP. At many promoters, CTD 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 CTD in RNAPCTD^I and CTD^IIcan 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 CTD 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 CTD that recruits CTD, 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-CTD 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-CTD 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 CTD indicates that the determinant of CTD responsible for CRP-CTD interaction is an ~20 × 10 Å surface located adjacent to the DNA-binding motif of CTD (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-CTD 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 CTD 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 CTD to the DNA segment immediately upstream of the DNA site for CRP (and also interacts with NTD; 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 CTD 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 CTD is available for potential interactions with a second molecule of CRP, or a molecule of another activator that functions by interacting with CTD (Belyaeva et al. 1998; Busby and Ebright 1999; Langdon and Hochschild 1999).

View larger version (47K): [in this window] [in a new window]   Figure 1.   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  C-terminal domain (CTD). (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 CTD, and (2) direct protein-protein interaction (grey filled circle) between AR2 in the downstream subunit of CRP and  subunit N-terminal domain (NTD).

The major aim of the work presented here has been to obtain direct evidence that although only one copy of CTD is required for CRP-dependent transcription at promoters having one DNA site for CRP, two copies of CTD 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  subunits or containing one full-length  subunit and one truncated  subunit lacking CTD.

A second aim of this work has been to address a long-standing point of contention, namely, whether a single copy of CTD is able to interact productively with both CRP and DNA. We have proposed that CTD 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 CTD 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 CTD that reduce CRP-dependent activation of the lac promoter, proposed that CTD contacts CRP and DNA through a single determinant (or two extensively overlapping determinants). Thus, one copy of CTD 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  subunit at a class II CRP-dependent promoter with a single UP-element subsite located immediately adjacent to the DNA site for CRP.

	Results

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

To determine the number of copies of CTD 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).

View larger version (20K): [in this window] [in a new window]   Figure 2.   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 subsitesone adjacent to DNA site for CRP, and one not adjacent to DNA site for CRP.

View larger version (43K): [in this window] [in a new window]   Figure 3.   Full activation at a cyclic AMP receptor protein (CRP)-dependent promoter having two DNA sites for CRP requires both copies of  C-terminal domain (CTD): 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  subunits (I/II) and with one full-length and one truncated  subunit (I/II). Yields of transcripts from CC(-61.5) and CC(-93.5)CC(-41.5) are normalized with reference to the control RNA I transcript.

We performed experiments using RNAP derivatives having two full-length  subunits, or one full-length  subunit and one truncated  subunit lacking CTD. We prepared RNAP derivatives by co-expressing, respectively, genes encoding , , `, , <sup>70</sup>, and hexahistidine-tagged , or genes encoding , , `, , ⁷⁰, and hexahistidine-tagged [RA45](1-235) (wherein the RA45 substitution prevents an  derivative from occupying the -associated ^I site within RNAP, and thereby restricts an  derivative to the `-associated ^II 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  subunits, or one full-length  subunit and one truncated  subunit lacking CTD. 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  subunit (Fig. 3, lanes 2,5), indicating that only one copy of CTD 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  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 CTD 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 CTD: class II promoter derivative having a second DNA site for CRP

To determine the number of copies of CTD 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).

View larger version (43K): [in this window] [in a new window]   Figure 4.   Full activation at a cyclic AMP receptor protein (CRP)-dependent promoter having two DNA sites for CRP requires both copies of  C-terminal domain (CTD): 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  subunits (I/II) and with one full-length and one truncated  subunit (I/II). 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  subunits, or one full-length -subunit and one truncated -subunit lacking CTD. 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  subunit (Fig. 4, lanes 2,5), indicating that only one copy of CTD 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  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 CTD 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 CTD

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

View larger version (41K): [in this window] [in a new window]   Figure 5.   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  C-terminal domain (CTD). Results of transcription experiments at (-59.5)CC(-41.5) (sequence in Fig. 2C) using Escherichia coli RNA polymerase holoenzyme (RNAP) derivatives with two full-length  subunits (I/II) and with one full-length and one truncated  subunit (I/II). Yields of transcripts from (-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 CTD

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

View larger version (45K): [in this window] [in a new window]   Figure 6.   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  C-terminal domain (CTD). Results of transcription experiments at CC(-69.5)(-51.5)(-41.5) (sequence in Fig. 2D) using Escherichia coli RNA polymerase holoenzyme (RNAP) derivatives with two full-length  subunits (I/II) and with one full-length and one truncated  subunit (I/II). Yields of transcripts from CC(-69.5)(-51.5)(-41.5) are normalized with reference to the control RNA I transcript.

View larger version (50K): [in this window] [in a new window]   Figure 7.   Models.  C-terminal domain (CTD),  subunit N-terminal domain (NTD), , ', , and  denote, respectively, the Escherichia coli RNA polymerase holoenzyme (RNAP)  subunit C-terminal domain, the RNAP  subunit N-terminal domain, and the RNAP , `, , and 70 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-CTD, AR2-NTD, and (UP-element subsite)-CTD interactions are indicated by, respectively, black filled circles, grey filled circles, and black filled bars. For clarity, copies of CTD 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 CTD not involved in AR1-CTD or (UP-element subsite)-CTD interactions are likely to make nonspecific DNA-CTD 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 CTD required) and at a derivative having a second DNA site for CRP (two copies of CTD required). (B) Transcription activation at a class II CRP-dependent promoter (one copy of CTD required) and at a derivative having a second DNA site for CRP (two copies of CTD required). (C) Transcription activation at a CRP-dependent promoter having one UP-element subsite adjacent to DNA site for CRP (one copy of CTD required). (D) Transcription activation at a CRP-dependent promoter having two UP-element subsitesone adjacent to DNA site for CRP, and one not adjacent to DNA site for CRP (two copies of CTD required).

	Discussion

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Our results establish that although only one copy of CTD 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 CTD 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 CTD 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 CTD 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 -associated  subunit, ^I, was full-length, and the `-associated  subunit, <sup>II</sup>, was truncated. Extensive data establish that CTD of <sup>I</sup> and CTD of <sup>II</sup> can function interchangeablyat 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 -associated  subunit, <sup>I</sup>, was truncated, and the `-associated  subunit, ^II, 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 CTD in RNAP (Fig. 7).

Our finding that a single CTD 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 CTD can interact productively with both CRP and DNA. This supports the proposal that CTD has distinct, nonoverlapping functional determinants for interaction with CRP and DNA, and that CTD 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 CTD interacts with both CRP and DNA (Murakami et al. 1996; Ishihama 1997; Ozoline et al. 2000). We suggest that CTD interacts simultaneously with activator and DNA in many, possibly all, activator-dependent, CTD-dependent transcription complexes.

	Materials and methods

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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.

To measure promoter activities, the fragments were cloned into the lac expression vector, pRW50 (Lodge et al. 1992), and -galactosidase expression in DH5 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). (-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  subunits (RNAP ^I/^II) and carrying one full-length  subunit and one truncated  subunit lacking CTD (RNAP ^I/^II) were prepared from transformants of E. coli strain XL1-blue with, respectively, pREII-NH and pREII-NH45A(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  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 CTD (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 -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).