Transcription Activation by CooA, the CO-sensing Factor from Rhodospirillum rubrum
THE INTERACTION BETWEEN CooA AND THE C-TERMINAL DOMAIN OF THE alpha  SUBUNIT OF RNA POLYMERASE*

Yiping He, Tamas Gaal, Russell Karls, Timothy J. Donohue, Richard L. Gourse, and Gary P. RobertsDagger

From the Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CooA, a member of the cAMP receptor protein (CRP) family, is a CO-sensing transcription activator from Rhodospirillum rubrum that binds specific DNA sequences in response to CO. The location of the CooA-binding sites relative to the start sites of transcription suggested that the CooA-dependent promoters are analogous to class II CRP-dependent promoters. In this study, we developed an in vivo CooA reporter system in Escherichia coli and an in vitro transcription assay using RNA polymerases (RNAP) from E. coli and from Rhodobacter sphaeroides to study the transcription properties of CooA and the protein-protein interaction between CooA and RNAP. The ability of CooA to activate CO-dependent transcription in vivo in heterologous backgrounds suggested that CooA is sufficient to direct RNAP to initiate transcription and that no other factors are required. This hypothesis was confirmed in vitro with purified CooA and purified RNAP. Use of a mutant form of E. coli RNAP with alpha  subunits lacking their C-terminal domain (alpha -CTD) dramatically decreased CooA-dependent transcription of the CooA-regulated R. rubrum promoter PcooF in vitro, which indicates that alpha -CTD plays an important role in this activation. DNase I footprinting analysis showed that CooA facilitates binding of wild-type RNAP, but not alpha -CTD-truncated RNAP, to PcooF. This facilitated binding provides evidence for a direct contact between CooA and alpha -CTD of RNAP during activation of transcription. Mapping the CooA-contact site in alpha -CTD suggests that CooA is similar but not identical to CRP in terms of its contact sites to the alpha -CTD at class II promoters.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CooA is a CO-sensing transcription activator from Rhodospirillum rubrum, and it activates the expression of the cooFSCTJ and cooMKLXUH operons in response to CO (1-3). These two adjacent operons encode proteins that oxidize CO to CO2 with concomitant reduction of H+ to H2 and allow growth of this organism on CO as a sole energy source (4-6). cooA lies immediately 3' of the cooFSCTJ operon, and its expression does not depend on CO.

cooA insertion and deletion mutants fail to accumulate mRNA from either the cooFSCTJ or cooMKLXUH operons in response to CO (1, 2). Sequence analysis suggested that CooA is a homolog of cAMP receptor protein (CRP),1 a well characterized transcription factor in Escherichia coli (1). The proposed helix-turn-helix DNA binding motif of CooA is highly similar to that of CRP, whereas the effector-binding domain displays lower similarity with CRP. CooA is not abundant in R. rubrum, but overexpression and purification of CooA were achieved in both R. rubrum and E. coli. CooA purified anaerobically from R. rubrum was indistinguishable spectroscopically or in terms of activity from CooA purified aerobically from either R. rubrum or E. coli (7, 8). CooA is homodimeric under all conditions and contains approximately 2 mol of protoheme/dimer. CO directly binds to the heme of the reduced form of CooA, but not to the oxidized form, and this CO binding induces a conformational change in CooA that allows it to bind DNA in a sequence-specific manner (8). Oxidized CooA can be readily reduced, acquiring the ability to bind CO, and the mechanism for this reversible redox reaction involves an unusual switch of protein ligands on one side of the heme (8, 9). Because the heme of CooA is 6-coordinate when reduced and when bound by CO, CO binding apparently displaces one of the axial ligands to the heme. It is our working hypothesis that this protein ligand displacement serves as the trigger for the conformational change in the CooA structure upon CO binding (8, 9).

E. coli RNAP is composed of alpha 2beta beta 'sigma subunits; the role of sigma  is to bind -10 and -35 promoter elements. At promoters with A + T-rich sequences upstream of the -35 hexamer, termed "UP elements," the C-terminal domain of the alpha  subunit (alpha -CTD) makes specific DNA contacts that enhance transcription initiation (13). Two regions of the alpha -CTD (including residues Leu262, Arg265, Asn268, Cys269, Gly296, Lys298, and Ser299) were found to be important for UP element-dependent transcription and DNA binding (14, 15). The alpha -CTD is an independently folding domain of RNAP that is joined to the N-terminal domain of the alpha  subunit by a flexible linker that allows the alpha -CTD to occupy different positions at different promoters (10-12). The alpha -CTD is also known to make specific contacts with a range of activator proteins (16, 17).

E. coli CRP controls the transcription of numerous genes involved in carbon source utilization. Upon the binding of its allosteric effector, cAMP, CRP undergoes a conformational change that allows a dimer of CRP to bind to a specific 22-base pair sequence at target promoters (consensus sequence is 5'-AAATGTGATCTAGATCACATTT-3', in which the most important bases for CRP recognition are in bold) and to activate transcription at those promoters (18). CRP-dependent promoters can be grouped into two classes based on the position of the CRP-binding site relative to the start of transcription as well as on the mechanism for transcription activation (19). At class I promoters, the DNA-binding site for CRP is upstream of that for RNAP and is centered at position -61.5, -71.5, -82.5, or -92.5. At class II CRP-dependent promoters, to which the CooA-dependent promoters are analogous, the binding site for CRP is centered at -41.5, overlapping the -35 region, and the alpha -CTD binds to DNA upstream of the CRP dimer.

Direct interaction between CRP and RNAP plays a pivotal role in transcription activation at both promoter classes (20, 21). In particular, transcription activation at class II promoters requires two distinct contacts between CRP and the alpha  subunit and a third contact between CRP and sigma 70. One interaction is between activating region 1 (AR1) of the upstream subunit of the CRP dimer and the alpha -CTD. This interaction increases initial binding of RNAP to the promoter (22). Recently, residues 285-288 and 317 of alpha -CTD have been shown to comprise the surface that interacts with AR1 of CRP at class II promoters (23). The second contact, between activating region 2 (AR2) of the downstream subunit of CRP and the N-terminal domain of the alpha  subunit, facilitates isomerization of the closed complex to the open complex (24). The residues in AR1 and AR2 of CRP are not conserved in CooA, which suggests that there might be certain differences in the interactions of CooA and CRP with alpha . The third activator region (AR3) in CRP, formed by residues 52-58, interacts with sigma 70 of RNAP (20). Because the AR3 region in CRP is highly similar to an analogous region in CooA, this region in CooA might serve an AR3-like function.

The two CooA-regulated R. rubrum promoters, PcooF and PcooM, contain 2-fold symmetric DNA sequences that serve as CooA-binding sites and are similar to the CRP consensus sequence. This is consistent with the similarity between CooA and CRP in their helix-turn-helix motifs (2, 3). The CooA-binding sites lie at the -43.5 and -38.5 positions relative to the transcription start sites in PcooF and PcooM, respectively, overlapping with the -35 region (2, 3). This overlap suggests that both CooA-regulated promoters are analogous to class II CRP-dependent promoters. We chose PcooF for this study because it is the stronger promoter based on the amount of primer extension product and level of coo-encoded proteins synthesized in vivo (1, 2).

Although CooA shares some common features with CRP, such as DNA binding properties and effector-induced activation, it displays striking differences from CRP in the effector-binding domain and in regions AR1 and AR2. We were interested to know whether CooA was necessary and sufficient for CO-dependent activation of transcription and whether the mechanism of activation by CooA was similar to that of CRP. In this work, we used in vivo CooA reporter systems and in vitro transcription assays to examine the properties of CooA in transcription activation. Because of the particular questions we wished to address, we performed the bulk of the work with RNAP from E. coli, so that any differences between CooA and the CRP detected would reflect properties of CooA. The nature of transcription activation by CooA was investigated through the study of the interaction between CooA and RNAP and, in particular, the interaction between CooA and the alpha -CTD.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of a System for CooA Expression and a Reporter of CooA Activity in E. coli-- CooA was overexpressed from a vector, pYHA1, created as follows. A 1.9-kilobase PvuI-BamHI fragment containing Ptac-cooA-rrnBT1T2, was isolated from pKK223-3 (8), digested with PvuI, mung bean nuclease, and BamHI, and cloned into the EcoRV and BamHI sites of pACYC184. A plasmid containing a PcooF-lacZ fusion, pYHF4, was constructed by inserting a polymerase chain reaction-amplified EcoRI-HindIII fragment, extending from positions -250 to +70 of PcooF, into plasmid pMSB1 (25), which created pMSBPcooF. The reporter region was recombined from plasmid pMSBPcooF into lambda  phage RS468 (26) in strain DH5alpha containing pYHA1. Lysogens were screened by the blue color of plaques on Luria broth (LB) + X-gal plates incubated anaerobically in the presence of CO. The promoter region of integrated PcooF-lacZ fusion in the chromosome was confirmed by DNA sequencing.

In Vitro Transcription Assays-- Polymerase chain reaction-amplified EcoRI-HindIII fragments from positions -250 to +70, -90 to +70, and -60 to +70 of PcooF were cloned into pRLG770, which contains transcription terminator rrnBT1T2 downstream of the multicloning site (27), to yield plasmids pYHF1, pYHF2, and pYHF3, respectively. The supercoiled plasmids used as DNA templates for these assays were purified with the Midi Kit from Qiagen. The RNAPs used in these experiments were Esigma 70 purified from E. coli or a Rhodobacter sphaeroides RNAP preparation enriched for the Esigma 70 homolog (28). Standard multiple-round transcription assays (13) were modified as described below to accommodate the requirement of CooA for an anoxic environment to bind CO (7). The sealed tubes containing 25-µl reactions (0.2 nM supercoiled plasmid, 3.5 nM RNAP, 40 nM CooA dimer, and a buffer (30 mM KCl, 40 mM Tris acetate, pH 7.9, 10 mM MgCl2, 1 mM dithiothreitol, 100 mg/ml bovine serum albumin, 200 mM ATP, 200 mM CTP, 200 mM GTP)) were degassed and filled with argon in the head space. After the addition of dithionite to 1.7 mM to scavenge any free oxygen, CO was added, and the reactions were incubated at room temperature for 15 min. This step served to activate CooA and allow the activated CooA to interact with the promoter and RNAP to form a predicted 21-nucleotide transcript by incorporating ATP, GTP, and CTP. The reactions were then exposed to air, and 10 mM UTP and 5 µCi of [32P]UTP (DuPont) were added to extend the mRNA at room temperature for 20 min. Reactions were terminated and electrophoresed as described (29). The signal intensities of transcripts were quantified using a PhosphorImager (Molecular Dynamics) and ImageQuant software.

DNase I Footprinting Assays-- A DNA fragment containing the PcooF sequence from position -90 to +70 was polymerase chain reaction-amplified with an unlabeled bottom strand primer and a top strand primer labeled with [gamma -32P]ATP and polynucleotide kinase. The amplified fragment was purified by polyacrylamide gel electrophoresis, followed by an Elutip Minicolumn (Schleicher & Schuell). The labeled fragment was incubated with CooA, or RNAP, or both in the presence of CO and under the stringent anoxic conditions described previously (2), except that 40 nM pure CooA and 3.5 nM RNAP were used in a 20-µl binding reaction. The reactions were treated with 2 units/ml RQ RNase-free DNase I (Promega) for 30 s. DNase I cleavage products were separated on a 6% (w/v) polyacrylamide-urea gel. Neither heparin nor any nucleotides were added into the reactions.

CO Induction of PcooF Expression in E. coli and Measurement of beta -Galactosidase Activity in Vivo-- Strains with a PcooF-lacZ reporter were grown aerobically in LB medium containing 100 mg/ml ampicillin and 35 mg/ml chloramphenicol for 12 h to reach stationary phase. In stoppered test tubes, 20-µl cultures were diluted into 2 ml of LB medium supplemented with 20 mM glucose and the same antibiotics. The air in the head space was replaced by argon and 2% CO, and the cultures were grown anaerobically to an A600 of approximately 0.45. beta -Galactosidase activity was determined according to Miller (30).

Protein Purification and Reconstitution of RNAP-- N-terminal His-tagged wild-type and mutant alpha  subunits were overexpressed from plasmid pHTT7f1-NHalpha (31) or derivatives constructed by gene replacement of the EcoRI-BamHI fragment with fragments encoding the desired alanine substitutions (14). Purification of alpha  subunits by Ni2+ affinity chromatography was performed as described in Tang et al. (31). Preparation of inclusion bodies of beta , beta ', and sigma 70 from strains XL1-Blue (pMKSe2), BL21 DE3 (pT7beta '), and BL21 DE3 (pLHN12 alpha ), respectively, and reconstitution of RNAP were carried out as described (31). CooA was purified from an overproducing strain of R. rubrum (UQ459) by the method of Shelver et al. (7).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CooA Is Necessary for CO-dependent Expression in Different Organisms-- CooA has been shown to be necessary for the CO-dependent expression of the two coo operons of R. rubrum (1, 2), and purified CooA binds DNA in a CO-dependent manner (7). These data are consistent with the hypothesis that CooA is both necessary and sufficient for sensing and activating transcription in response to CO, but they are not conclusive. We therefore examined the requirement for CooA in two heterologous systems. We chose R. sphaeroides because it is related to R. rubrum, yet does not appear to have the coo system, as judged by the absence of CO dehydrogenase activity and a failure to hybridize to probes of the coo genes (2). In this organism, a pRK404-based plasmid carrying cooFSCTJ with its normal promoter failed to produce detectable CO dehydrogenase activity (the product of cooS) in response to CO. However, when cooA was added to the plasmid in its normal position at the 3' end of the cooFSCTJ operon, exposure of the R. sphaeroides strain carrying this plasmid to CO produced easily detectable CO dehydrogenase activity (2).

We then examined CO- and CooA-dependent transcription in E. coli, an organism that is less related to R. rubrum and also lacks any evidence of a coo system. For this test, a reporter system was constructed that contained a PcooF-lacZ fusion in the chromosome and a plasmid overexpressing CooA. In this system, we detected a substantial increase of beta -galactosidase activity upon CO induction (200 Miller units in the presence of CO and 1.6 units in its absence), suggesting that CooA is sufficient for activating the transcription of PcooF in E. coli. Similar results with CooA reporters in E. coli have recently been reported by others (9). These results establish that CooA is necessary for the transcriptional response to CO and that it is able to associate productively in vivo with RNAPs from both E. coli and R. sphaeroides.

CooA Is Sufficient to Activate the Transcription of PcooF in Vitro-- The above results indicate that CooA is necessary, but only in vitro analysis can establish whether it is sufficient for CO-dependent transcriptional activation or whether additional factors are required for this activation. The ability of CooA to activate PcooF was studied by monitoring RNA synthesis in a purified system containing only DNA, RNAP, CooA, nucleotide triphosphates, and the proper buffer. To investigate the nature of CooA-mediated activation at the PcooF promoter, we modified the standard in vitro transcription assay, because CooA is only able to bind CO when reduced. The modified assay, detailed under "Experimental Procedures," was kept anoxic in the presence of CO to support activation by CooA until the formation of a 21-nucleotide transcript from PcooF. At this point, the reaction was exposed to air and extended aerobically for technical convenience. When the reactions were maintained strictly anoxically throughout the entire experiment, a quantitatively similar result was obtained, indicating that the in vitro transcription assay conditions we employed were sufficient for maximal CooA activity (data not shown).

The in vitro transcription assays were performed with a supercoiled DNA template (pYHF1) containing the PcooF and extending -250 base pairs upstream and +70 base pairs downstream of transcription start site. The reactions were carried out in the presence or absence of CO. As shown in lanes 2 and 4 of Fig. 1, CO-dependent transcripts were detected using RNAP from both R. sphaeroides and E. coli. The observed size of the transcript from PcooF correlated well with the predicted size of 240 nucleotides. In the absence of CO, no transcripts from PcooF were seen, whereas transcription of the control (RNA-1) was not affected by CO (Fig. 1, lanes 1 and 3).


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Fig. 1.   In vitro transcription of CooA with wild-type or alpha -CTD-truncated RNAPs. A supercoiled DNA template containing PcooF sequences from -250 to +70 of the transcription start site was used in this assay. The RNAPs used in each pair of reactions are wild-type (WT) RNAP from E. coli and R. sphaeroides (R. s) and the mutant E. coli RNAP with a truncation of the C terminus of alpha  starting at residue 235 (alpha -235). The + and - on the line labeled "CO" refer to the presence or the absence of CO in the headspace of the reaction tubes. The positions of transcripts initiated at the PcooF and the RNA-1 promoter are indicated by arrows.

These results demonstrate that CooA is sufficient for CO-dependent transcriptional activation and that no other factors are required. The results with the Esigma 70 from E. coli indicate that PcooF can be recognized by sigma 70 when activated by CooA.

CooA-activated Transcription Requires the alpha -CTD of RNAP-- Because the alpha -CTD makes specific DNA contacts at some promoters and specific protein contacts with a number of transcription activators including the CooA homolog CRP (13, 20), we wished to test whether there were similar contacts between alpha -CTD and either CooA or PcooF. We first examined whether the C-terminal domain of the RNAP alpha  subunit is required for transcription activation of PcooF by CooA. To address this question, we assayed the ability of a reconstituted Esigma 70 containing a truncation of the C-terminal domain of alpha  to direct transcription from CooA-dependent promoter PcooF in vitro. Use of E. coli RNAP with the alpha -CTD truncation resulted in a dramatic reduction of transcription from PcooF (Fig. 1, lanes 5 and 6). The enzyme activities of wild-type and mutant RNAPs were similar as demonstrated by transcription from the RNA-1 promoter. With a longer exposure of the x-ray film, we were able to detect a low level of a CO-dependent transcript from PcooF using RNAP with alpha -235 (data not shown). Because the alpha -CTD is known to make contacts with activators and UP elements, the ineffectiveness of the alpha -CTD-truncated RNAP for PcooF transcription suggests that the alpha  truncation disrupts the binding of alpha  to CooA, to a UP element, or to both.

CooA Utilizes Protein-Protein Contacts with alpha -CTD to Facilitate RNAP Binding to PcooF-- To analyze potential protein-protein interaction between CooA and RNAP and to specifically address interactions between alpha -CTD and CooA, we performed DNase I footprinting experiments using wild-type RNAP or the alpha -CTD-truncated RNAP in the presence or absence of CooA. As shown in Fig. 2, lanes 2-4, CooA alone protected PcooF on the top strand from positions -55 to -31 relative to the start site of transcription, in agreement with our previous observations (2, 7). In contrast, neither the wild-type nor the alpha -CTD-truncated RNAPs alone protected PcooF from DNase I (lanes 5 and 7). This result indicates that RNAP does not form a stable complex at PcooF in the absence of CooA. When both CooA and wild-type RNAP were incubated together with the DNA fragment, the protected region extended from the CooA-binding site in both directions, upstream to -68 and downstream to at least -1 (lanes 8 and 9). A similar result was obtained when R. sphaeroides RNAP was used, indicating that the RNAPs from these two different organisms function similarly in interacting with CooA at PcooF (lanes 10-12). In contrast to the results obtained with the wild-type RNAP, the alpha -CTD-truncated E. coli RNAP showed no evidence of forming a stable complex even in the presence of CooA (lane 6). This suggests that CooA activates transcription by enhancing the initial and stable binding of RNAP to PcooF through direct protein-protein contact with the alpha -CTD.


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Fig. 2.   DNase I footprinting analysis of CooA interacting with RNAP. A 3'-32P-end-labeled polymerase chain reaction fragment containing PcooF from position -90 to +70 was incubated with various concentrations of CooA and 3.5 nM RNAP in the presence of CO and under stringent anoxic conditions. The filled bar on the right indicates the region protected by CooA, whereas the open plus the filled bar represents the region protected by wild-type (WT) RNAP plus CooA. Lane 1, no protein; lanes 2-4, CooA protein; lane 5, the RNAP variant with the alpha -CTD truncation (alpha -235); lane 6, CooA plus the RNAP variant with the alpha -CTD truncation; lane 7, wild-type RNAP from E. coli; lanes 8 and 9, CooA plus wild-type RNAP from E. coli; lane 10, wild-type RNAP from R. sphaeroides (R. s); lanes 11 and 12, CooA plus wild-type RNAP from R. sphaeroides.

Analysis of CooA-regulated Promoter PcooF-- As noted above, the presence of CooA and wild-type RNAP, but not the alpha -CTD-truncated variants, caused DNase I protection to extend upstream and downstream of that region protected by CooA alone. The region upstream of the CooA-binding site is A + T-rich (Fig. 3) relative to most of the R. rubrum genome, reminiscent of the A + T-richness of UP elements in E. coli promoters that contact the alpha -CTD to increase transcription (13, 38). To test whether alpha  interacts with this region in a sequence-specific manner, we created PcooF constructs differing only in the extent of that region. PcooF sequences from positions -90 to +70 and -60 to +70 were cloned into the transcription assay vector pRLG770, resulting in the constructs pYHF2 and pYHF3, respectively. These plasmids were tested for CooA-dependent in vitro transcription activity with wild-type RNAP. The level of transcripts from those constructs was quantitatively compared with that of the construct pYHF1 containing the PcooF sequences from -250 to +70. As shown in Fig. 4, these three templates yielded similar amounts of CooA-dependent transcripts, indicating that the specific sequences upstream of position -60 in PcooF do not contribute significantly to promoter activity. This also suggests that the upstream A + T-rich sequence of PcooF does not act as a UP element. Although we cannot exclude the possibility that some portion of the protection upstream of the CooA-binding site is due to a conformational change in CooA induced by the presence of RNAP, the interaction of alpha -CTD with DNA upstream of CooA is consistent with the sequence-nonspecific interactions between DNA and alpha -CTD observed at class II CRP-dependent promoters (23).


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Fig. 3.   Sequence of PcooF region. The figure shows the upper strand sequence of PcooF. The transcription start is at +1. The underlined sequences represent the putative -10 region, the 2-fold symmetrical CooA-binding site, and the A + T-rich sequence upstream of CooA-binding site as marked, respectively. The region protected by CooA in DNase I footprinting experiments is indicated by the filled bar. The open bar represents the region protected by wild-type RNAP plus CooA.


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Fig. 4.   In vitro transcription analysis of PcooF deletions. The transcription reactions were performed in the same manner as described in Fig. 1. All of the reactions contained wild-type RNAP from E. coli, CooA, and CO. The supercoiled DNA templates used in the assay differ only in the extent of the upstream sequence of PcooF. Lane 1, pYHF3 containing PcooF sequences from -60 to +70; lane 2, pYHF2 containing PcooF sequences from -90 to +70; lane 3, pYHF1 containing PcooF sequences from -250 to +70. The positions of transcripts initiated at the PcooF and the RNA-1 promoter are indicated by arrows.

Identification of Residues in alpha -CTD That are Critical for CooA-dependent Activation at PcooF-- We wished to determine whether CooA made similar contacts with the alpha -CTD as seen with its homolog, CRP, and also to test whether the extended DNase I protection upstream of CooA reflected the direct contact of alpha -CTD with DNA. To address these questions, we measured CooA-activated transcription using a CooA reporter system containing a PcooF-lacZ fusion integrated into the E. coli chromosome, a plasmid expressing CooA, and a library of plasmids encoding single alanine substitutions throughout the entire alpha -CTD (23). The effects of the alpha -CTD mutants in this screen were small and somewhat variable (data not shown), but they did identify a few potential mutants that were then assayed for their effects on CooA-dependent transcription in vitro.

We tested reconstituted RNAPs in vitro containing single alanine substitutions for Thr285, Val287, Glu288, and Arg317 (the patch on alpha  that interacts with CRP at class II CRP-dependent promoters (23)), Arg265 (the residue most important for interaction of alpha  with DNA (14)), and a few additional mutants suggested by the in vivo screen, including Val306, Leu307, and Ser313.

The results of the in vitro transcription analysis with a subset of the variant RNAPs are shown in Fig. 5. The activity of each RNAP preparation was normalized to the transcription from the RNA-1 promoter. The R265A, L307A, and V287A RNAPs were defective in CooA-dependent transcription of PcooF, providing 13, 34, and 37% of wild-type RNAP activity, respectively (Fig. 5, lanes 2, 3, and 7). Because R265A (14) and L307A2 affected UP element-dependent transcription and RNAP extended the DNase I protection upstream of the CooA-binding site, this strongly suggested that alpha -CTD makes contacts with DNA upstream of CooA and that these contacts are important for CooA-meditated transcription activation. V287A was also defective in CooA-dependent transcription, although the other alpha -CTD variants important for class II CRP-dependent transcription (T285A, E288A, and R317A) had little or no effect. These results suggest that the CooA contact site in the alpha -CTD of RNAP shares some determinants of, but is not identical to, the site for CRP contact at class II-type promoters. RNAPs containing alpha  mutants V306A and S313A, which were also suggested by the in vivo screen, were not defective in CooA-dependent transcription in vitro (data not shown).


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Fig. 5.   In vitro transcription analysis of alpha -CTD variants. The E. coli RNAPs were reconstituted with either wild-type (WT) alpha  subunit or the alpha  subunits containing alanine substitutions at the positions indicated in the figure. All of the reactions were carried out in the presence of CooA, CO, and the supercoiled DNA template with PcooF sequences from -250 to +70. The positions of transcripts initiated at PcooF and the RNA-1 promoter are indicated by arrows. Based on the quantitation of transcript accumulations, R265A, L307A, and V287A show 13, 34, and 37% of wild-type RNAP activity for PcooF, respectively. These percentages represent the averages of three independent experiments in which all values were within 18% of the average.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Many transcription activators directly contact the alpha  subunit of RNAP (16, 17). In this study, we determined that CooA is sufficient for directing RNAP to initiate transcription of PcooF and that CooA-activated transcription of PcooF requires the C-terminal domain of the RNAP alpha  subunit. Consistent with these observations, CooA facilitates the binding of wild-type RNAP to PcooF but not that of alpha -CTD-truncated RNAP. These observations suggest that direct protein-protein contact between CooA and the alpha -CTD of RNAP plays an essential role in transcription activation of PcooF.

We suspect that the alpha -CTD/CooA interaction plays a role similar to that between AR1 of CRP and alpha -CTD at class II CRP-dependent promoters. In class II CRP-dependent promoters, alpha -CTD makes nonspecific contacts with the DNA segment immediately upstream of the CRP site (16, 23). In this study, we found that wild-type RNAP, but not RNAP with the alpha -CTD truncation, extends DNase I protection upstream of that protected by CooA alone. Furthermore, we determined that alpha  R265A and L307A, which decrease UP element-dependent transcription, also affect PcooF activity. Because the specific DNA sequence upstream of the CooA-binding site is not critical for PcooF activity, we propose that alpha -CTD makes nonspecific contacts with the DNA immediately upstream of the CooA site and that this protein-DNA interaction is important for CooA-activated transcription of PcooF.

Val287 is also important for CooA-activated transcription, although the other tested alpha  residues that contact CRP at class II promoters are not critical for CooA-dependent transcription of PcooF. Therefore, a different set of side chains, but probably the same region of alpha -CTD, might be important for activation by CRP and CooA. This hypothesis is consistent with the low similarity between CRP and CooA in activating region 1 (22).

In the DNase I footprinting analysis, RNAP does not completely protect the DNA downstream of PcooF +1, even in the presence of CooA and in the absence of heparin (Fig. 2). In addition, the majority of the CooA-RNAP-PcooF complexes detected in gel-shift assays are heparin-sensitive (data not shown). This partial downstream protection and the heparin sensitivity suggest that the detected ternary complex (CooA-RNAP-PcooF) may be a mixed population of closed and open complexes. Although most CRP-dependent promoters, such as lacP1, melT, galP1, form stable open complexes with RNAP in the presence of CRP (32-35), other promoters, such as rrnB P1 even in the presence of its activator protein Fis (36), form an unstable open complex with RNAP (37).

Because basal level transcription of PcooF in the absence of active CooA is not detectable, it is formally possible that the deletion of alpha -CTD affects basal level transcription of PcooF and not its activation by CooA. However, the requirement for at least one residue in alpha -CTD (Val287) that has no effect on DNA binding (23) suggests that the alpha -CTD requirement involves, at least in part, a protein-protein interaction between CooA and alpha -CTD.

    ACKNOWLEDGEMENTS

We thank Daniel Shelver for providing pure CooA and Marcin Filutowicz for suggestions and for reading the manuscript.

    FOOTNOTES

* This work was supported by the College of Agricultural and Life Science and by National Institutes of Health Grants GM 53228 (to G. P. R.), GM 37048 (to R. L. G.), and GM 37509 (to T. J. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Bacteriology, University of Wisconsin, 106A E. B. Fred Hall, 1550 Linden Dr., Madison, WI 53706-1567; Tel.: 608-262-3567; Fax: 608-262-9865; E-mail: groberts{at}bact.wisc.edu.

2 S. Bales, M. Burgess, S. Aiyar, and R. L. Gourse, unpublished data.

    ABBREVIATIONS

The abbreviations used are: CRP, cAMP receptor protein; RNAP, RNA polymerase; alpha -CTD, the C-terminal domain of the alpha  subunit; AR1, activating region 1; AR2, activating region 2; AR3, activating region 3; UP elements, A + T-rich sequences located upstream in certain promoters that increase promoter strength; X-gal, 5-bromo-4-chloro-3-indolylbeta -D-galactopyranoside.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Shelver, D., Kerby, R. L., He, Y., and Roberts, G. P. (1995) J. Bacteriol. 177, 2157-2163[Abstract]
  2. He, Y., Shelver, D., Kerby, R. L., and Roberts, G. P. (1996) J. Biol. Chem. 271, 120-123[Abstract/Free Full Text]
  3. Fox, J. D., He, Y., Shelver, D., Roberts, G. P., and Ludden, P. W. (1996) J. Bacteriol. 178, 6200-6208[Abstract]
  4. Bonam, D., Lehman, L., Roberts, G. P., and Ludden, P. W. (1989) J. Bacteriol. 171, 3102-3107[Medline] [Order article via Infotrieve]
  5. Kerby, R. L., Hong, S. S., Ensign, S. A., Coppoc, L. J., Ludden, P. W., and Roberts, G. P. (1992) J. Bacteriol. 174, 5284-5294[Abstract]
  6. Kerby, R. L., Ludden, P. W., and Roberts, G. P. (1995) J. Bacteriol. 177, 2241-2244[Abstract]
  7. Shelver, D., Kerby, R. L., He, Y., and Roberts, G. P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11216-11220[Abstract/Free Full Text]
  8. Shelver, D., Reynolds, M. F., Thorsteinsson, M. V., Kerby, R. L., Chung, S. Y., Parks, R. B., Burstyn, J. N., and Roberts, G. P. (1999) Biochemistry 38, 2669-2678[CrossRef][Medline] [Order article via Infotrieve]
  9. Aono, S., Ohkubo, K., Matsuo, T., and Nakajima, H. (1998) J. Biol. Chem. 273, 25757-25764[Abstract/Free Full Text]
  10. Blatter, E., Ross, W., Tang, H., Gourse, R. L., and Ebright, R. (1994) Cell 78, 889-896[Medline] [Order article via Infotrieve]
  11. Busby, S., and Ebright, R. H. (1994) Cell 79, 743-746[Medline] [Order article via Infotrieve]
  12. Jeon, Y. H., Negishi, T., Shirakawa, M., Yamazaki, T., Fujita, N., Ishihama, A., and Kyogoku, Y. (1995) Science 270, 1495-1497[Abstract]
  13. Ross, W., Gosink, K. K., Salomon, J., Igarashi, K., Zou, C., Ishihama, A., Severinov, K., and Gourse, R. L. (1993) Science 262, 1407-1413[Medline] [Order article via Infotrieve]
  14. Gaal, T., Ross, W., Blatter, E., Tang, H., Jia, X., Krishnan, V. V., Assa-Munt, N., Ebright, R. H., and Gourse, R. L. (1996) Genes Dev. 10, 16-26[Abstract]
  15. Murakami, K., Fujita, N., and Ishihama, A. (1996) EMBO J. 15, 4358-4367[Abstract]
  16. Ebright, R. H., and Busby, S. (1995) Curr. Opin. Genet. Dev. 5, 197-203[CrossRef][Medline] [Order article via Infotrieve]
  17. Ishihama, A. (1992) Mol. Microbiol. 6, 3283-3288[Medline] [Order article via Infotrieve]
  18. Kolb, A., Busby, S., Buc, H., Garges, S., and Adhya, S. (1993) Annu. Rev. Biochem. 62, 749-795[CrossRef][Medline] [Order article via Infotrieve]
  19. Ebright, R. (1993) Mol. Microbiol. 8, 797-802[Medline] [Order article via Infotrieve]
  20. Busby, S., and Ebright, R. H. (1997) Mol. Microbiol. 23, 853-859[Medline] [Order article via Infotrieve]
  21. Dove, S. L., Joung, J. K., and Hochschild, A. (1997) Nature 386, 627-630[CrossRef][Medline] [Order article via Infotrieve]
  22. Zhou, Y., Pendergrast, P. S., Bell, A., Williams, R., Busby, S., and Ebright, R. (1994) EMBO J. 13, 4549-4557[Abstract]
  23. Savery, N. J., Lloyd, G. S., Kainz, M., Gaal, T., Ross, W., Ebright, R. H., Gourse, R. L., and Busby, S. (1998) EMBO J. 17, 3439-3447[Abstract/Free Full Text]
  24. Niu, W., Kim, Y., Tau, G., Heyduk, T., and Ebright, R. (1996) Cell 87, 1123-1134[Medline] [Order article via Infotrieve]
  25. Rao, L., Ross, W., Appleman, J. A., Gaal, T., Leirmo, S., Schlax, P. J., Record, M. T., and Gourse, R. L. (1994) J. Mol. Biol. 235, 1421-1435[CrossRef][Medline] [Order article via Infotrieve]
  26. Simons, R. W., Houman, F., and Kleckner, N. (1987) Gene 53, 85-96[CrossRef][Medline] [Order article via Infotrieve]
  27. Ross, W., Thompson, J. F., Newlands, J. T., and Gourse, R. L. (1990) EMBO J. 9, 3733-3742[Abstract]
  28. Karls, R., and Donohue, T. J. (1993) J. Bacteriol. 175, 7629-7638[Abstract]
  29. Leirmo, S., and Gourse, R. L. (1991) J. Mol. Biol. 220, 550-568
  30. Miller, J. H. (1992) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  31. Tang, H., Severinov, K., Goldfarb, A., and Ebright, R. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4902-4906[Abstract]
  32. Tagami, H., and Aiba, H. (1995) Nucleic Acids Res. 23, 599-605[Abstract]
  33. Eichenberger, P., Dethiollaz, Z., Fujiti, N., Ishihama, A., and Geiselmann, J. (1996) Biochemistry 35, 15302-15312[CrossRef][Medline] [Order article via Infotrieve]
  34. Herbert, M., Kolb, A., and Buc, H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2807-2811[Abstract]
  35. Tagami, H., and Aiba, H. (1998) EMBO J. 17, 1759-1768[Abstract/Free Full Text]
  36. Bokal, A. J., Ross, W., Gaal, T., Johnson, R. C., and Gourse, R. L. (1997) EMBO J. 16, 154-162[Abstract/Free Full Text]
  37. Gaal, T., Bartlett, M. S., Ross, W., Turnbough, C. L., and Gourse, R. L. (1997) Science 278, 2092-2097[Abstract/Free Full Text]


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