©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence for Contact between the Cyclic AMP Receptor Protein and the Subunit of Escherichia coli RNA Polymerase (*)

(Received for publication, April 17, 1995)

Ruzhong Jin (§) Karim A. Sharif Joseph S. Krakow (¶)

From the Department of Biological Sciences, Hunter College of the City University of New York, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The loop at the 52-position of the cAMP receptor protein (CRP) has been suggested as a potential site for contacting RNA polymerase on Class II promoters where the CRP binding site is located at position -41.5 (Bell, A., Gaston, K., Williams, R., Chapman, K., Kolb, A., Buc, H., Minchin, S., Williams, J., and Busby, S.(1990) Nucleic Acids Res. 18, 7243-7250). Using protein-protein photo-cross-linking, evidence is presented showing that the 52-loop of CRP is in physical proximity to the subunit of RNA polymerase holoenzyme. This interaction required the presence of a functional preinitiation complex. The CRP suppressor mutation, K52N, increased the efficiency of cross-linking, indicating an improved physical interaction between the CRP 52-loop and the subunit. Evidence for direct interaction between the CRP 156-162 loop and alpha subunit of RNA polymerase on both gal and lac promoters are also provided. The data indicate that CRP bound to the gal promoter contacts both the alpha and subunits of RNA polymerase.


INTRODUCTION

CRP (^1)is a dimeric protein which binds to consensus sites on the promoter through a helix-turn-helix motif in its C-terminal domain. The CRP target sites on DNA are located at a variety of positions with respect to the transcription initiation site. Typically, Class I promoters (like lac P1) have the CRP binding site at position -61.5, while that of Class II promoters (like gal P1) is located at position -41.5.

Evidence indicates that contact between CRP and RNA polymerase is involved in transcription activation at the lac promoter. DNase I footprinting demonstrated that CRP and RNA polymerase cooperatively bind to the lac promoter (Li and Krakow, 1985; Ren et al., 1988; Straney et al., 1989). Fluorescence polarization experiments showed that CRP and RNA polymerase interact in solution (Heyduk et al., 1993). CRP ``positive control'' mutants which affect transcription activation without altering specific DNA binding and bending have been isolated; they map on an exposed loop spanning amino acid residues 156 to 162 in the C-terminal domain (Bell et al., 1990; Eschenlauer et al., 1991; Zhou et al., 1993). C-terminal truncated mutants of the alpha subunit of RNA polymerase cannot be activated by CRP on the lac promoter (Igarashi et al., 1991; Kolb et al., 1993). Sharif et al. (1994) showed that monoclonal antibodies directed against the alpha subunit inhibited transcription from the CRP-dependent lac P1 promoter but not from the lac UV5 and gal promoters. CRP activates transcription from Class I promoters by contact of a primary activation site in its C-terminal domain (amino acid residues 156-162) with the C terminus of the alpha subunit of RNA polymerase. Protein-protein photo-cross-linking has provided direct evidence that the CRP 161 position contacts the C-terminal domain of the alpha subunit of RNA polymerase (Chen et al., 1994).

The mechanism of transcription activation on Class II promoters is less clear. Evidence suggests that the contact site for CRP on Class II promoters differs from those on Class I promoters. Mutations in the alpha subunit of RNA polymerase abolish CRP activation for Class I promoters with no effect on Class II promoters (Igarashi et al., 1991; Igarashi and Ishihama, 1991; Zou et al., 1992). C-terminal truncations in the subunit impair the ability of RNA polymerase to respond to CRP activation for gal transcription, suggesting that the RNA polymerase activation site for CRP on a Class II promoter might be located within the deleted C-terminal domain of (Kumar et al., 1994). Point mutations at position 52 (K52N,K52Q) restore the CRP stimulatory activity of 156-162 pc mutants (H159L, G162C, Delta159) on the gal promoter but not on lac (Bell et al., 1990; Williams et al., 1991). These observations suggest that there are at least two different sites on CRP which contact RNA polymerase at two different positions depending upon the promoter class involved. Numerous studies of the lac promoter show that the surface-exposed CRP loop consisting of amino acids 156-162 (Activating Region I) makes direct contact with a site on the C terminus of the RNA polymerase alpha subunit. In a recent study, Busby and his collaborators (Attey et al., 1994) found that alpha protects upstream sequences both in the binary RNA polymerasebulletgal P1 and ternary RNA polymerasebulletCRPbulletgal P1 complexes. In the latter complexes, alpha appears to make direct contact with the Activating Region I, spanning amino acids 156-162, of CRP.

In this report, we provide evidence that CRP bound to the gal promoter contacts both the alpha and subunits of RNA polymerase.


MATERIALS AND METHODS

Reagents

Reagents were obtained as follows. cAMP and benzophenone-4-maleimide, Sigma; restriction endonucleases, T4 DNA ligase and Vent DNA polymerase, New England Biolabs; Sequenase Version 2.0, U. S. Biochemical Corp.; DNase I, Promega; ECL Western blotting analysis system, Amersham. Anti- monoclonal antibody 2D1 (Strickland et al., 1988) was from Dr. Richard Burgess. Anti-CRP monoclonal antibody 64D1 (Li and Krakow, 1985), anti-alpha monoclonal antibody 23C2 (Sharif et al., 1994), and anti-beta and anti-beta` monoclonal antibodies (Riftina et al., 1989; Rockwell and Krakow, 1988) were prepared in this laboratory.

Bacterial Strains and Plasmids

Escherichia coli M182 (Deltacrp39, Deltalac), plasmids pRW2, promoters CCpmelR and CC+20pmelR (Gaston et al., 1990) were from Dr. Stephen Busby. A crp strain, XE64.2, was obtained from Dr. Richard Ebright.

PCR Site-specific Mutagenesis

Site-specific mutagenesis by the use of polymerase chain reaction was carried out on the crp gene according to the method of Ito et al. (1991). Three common primers and the primers specific for the indicated mutations were used in the PCR reaction.

beta-Galactosidase Assay

beta-Galactosidase assays were carried out in quadruplicate by the method of Miller(1972) using sodium dodecyl sulfate and chloroform. Cells were grown at 37 °C overnight in LB medium supplemented with 100 µg/ml ampicillin and 100 µM isopropyl-1-thio-beta-D-galactopyranoside. The overnight cultures were reinoculated into the same medium and grown at 37 °C until they reached midlog phase when they were used to determine beta-galactosidase activity.

Protein Purification

Mutant and wild-type CRPs were purified by a modification of the method of Eilen and Krakow(1977). RNA polymerase was isolated from E. coli K12 by a modification of the method of Burgess and Jendrisak(1975).

Preparation of BPM-CRP

Benzophenone-4-maleimide-labeled CRP (BPM-CRP) was prepared by a modification of the method of Tao et al.(1985). Mutant CRP (1-2 mg/ml) in CRP storage buffer (10 mM potassium phosphate (pH 7.0), 1 mM EDTA, 0.2 M NaCl, without dithiothreitol) was labeled by adding BPM from a 20 mM stock solution in dimethylformamide to a final BPM to CRP ratio of 4 to 1. The reaction was allowed to proceed overnight in the dark on ice, followed by quenching with excess dithiothreitol and dialysis against CRP storage buffer containing 0.1 mM dithiothreitol. Protein concentration was determined by using the Bio-Rad protein assay kit.

Photo-cross-linking

Cross-linking was carried out in a final volume of 50 µl containing 50 mM Tris-HCl (pH 7.8), 3 mM magnesium acetate, 0.1 mM EDTA, 0.1 mM dithiothreitol, 50 mM NaCl, 20 pmol of BPM-CRP, 20 pmol of the indicated promoter DNA fragment, 15 pmol of RNA polymerase holoenzyme with or without 0.1 mM cAMP. The mixture was incubated at 37 °C for 30 min and subjected to UV irradiation (>300 nm) for 30 min at room temperature followed by DNase I digestion (5 units) for 15 min at 37 °C.

Western Blot Assay

Proteins were separated on a SDS-polyacrylamide gel following the procedure of Laemmli(1970). The proteins were transferred at 2.5 mA/cm^2 for 45 min to a nitrocellulose membrane using a MiniBlot-SDE (Millipore). Identification of antigens on the nitrocellulose membrane was performed according to the method of Johnson et al.(1984). Due to the high molecular weight of beta, beta`, and the cross-linked product, Pronase was used to facilitate transfer to the nitrocellulose membrane (Gibson, 1981).


RESULTS

CRP stimulates transcription on a Class I promoter by contact of its activating region at the 156-162 loop with the C-terminal region of the alpha subunit of RNA polymerase. Another surface-exposed loop at position 52 on CRP is also considered a potential candidate for contacting RNA polymerase during Class II promoter activation. To directly study possible contact with RNA polymerase, site specific photo-cross-linking was carried out using benzophenone-4-maleimide as a sulfhydryl-specific photoreactive cross-linker.

The reactive cysteine 178 of CRP was first replaced with serine (Pendergrast et al., 1992), and a new cysteine was introduced into the CRP 52-loop at position 56. Another mutant was constructed having the K52N substitution in addition to its G56C substitution. The two CRP mutants constructed for photo-cross-linking were C178S,G56C and C178S,K52N,G56C. Transcription activation was determined in vivo by assaying beta-galactosidase activity. Both mutant CRPs retain the ability to activate transcription on both lac and gal type promoters (Fig. 1). The mutant CRPs were purified and reacted with benzophenone-4-maleimide (BPM) to specifically modify the solvent-accessible cysteine residue on CRP introduced by site-directed mutagenesis into the 52-loop at the 56-position.


Figure 1: A, in vivo expression of the lac operon. beta-Galactosidase assays show the stimulating activity of mutant and wild-type CRPs which were expressed in E. coli XE64.2 cells. B, in vivo expression of the gal-like class II promoter CCpmelR. beta-Galactosidase assays show the stimulating activity of mutant and wild-type CRPs which were expressed in E. coli M182 cells (Deltacrp39,Deltalac) with the co-transformation of CCpmelR promoter in pRW2.



To assess the ability of the BPM-CRP to contact RNA polymerase, photo-cross-linking was carried out on the (BPM-CRP)bulletDNAbulletRNA polymerase ternary complex. After UV irradiation, DNase I was added to digest the promoter DNA, and the mixture was resolved on a SDS-10% polyacrylamide gel. Western blotting with monoclonal antibodies specific for CRP, alpha, beta, beta`, and was carried out to detect possible cross-linked products. The results indicated that BPM-CRP cross-linked with only the subunit of RNA polymerase via the CRP N-terminal 52-loop. After photoactivation in the presence of cAMP and promoter DNA, a novel protein band with a molecular mass of approximately 100 kDa was observed on the Western blot which cross-reacted with both the anti-CRP (64D1) and anti- (2D1) monoclonal antibodies (Fig. 2). Several bands which cluster around 45 kDa were also produced in the presence or absence of cAMP. Since these are recognized only by anti-CRP antibodies and are produced even in the absence of RNA polymerase (results not shown), we believe they are cross-linked CRP monomers. No cross-linking of CRP with alpha, beta, or beta` was detected (results not shown).


Figure 2: Western blotting experiments showing the photo-cross-linking results using the BPM-derivatized mutant CRPs on CCpmelR promoter DNA fragment. The CCpmelR DNA is 186 bp containing the promoter region from -90 to +96; the nonspecific DNA is 242 bp containing the C-terminal sequence of the crp gene from +481 plus a small sequence of the adjacent pBR322 vector. The indicated DNA fragments were produced by PCR. A, BPM-CRP C178S,G56C was used for photo-cross-linking. B, BPM-CRP C178S,K52N,G56C was used for photo-cross-linking.



The photo-cross-linking between the CRP 52-loop and subunit of the RNA polymerase holoenzyme is cAMP-dependent in the presence of promoter DNA. In the absence of cAMP, the cross-linked product of 100 kDa was not detectable, whereas the cross-linked CRP dimers at 45 kDa were still present. In the presence of cAMP and the class II promoter DNA CCpmelR, both BPM-CRPs (C178S,G56C and C178S,K52N,G56C) cross-linked with the subunit (Fig. 2). The mutant BPM-CRPs were able to cross-link with on a class I promoter CC+20pmelR but with lower efficiency. The cross-linking efficiencies of the CRP derivatives on the gal-like CCpmelR or lac-like CC+20pmelR with are better than on the natural gal and lac promoters, presumably because of the consensus CRP binding sites on these synthetic promoters (Fig. 3).


Figure 3: Western blotting experiment showing the photo-cross-linking results for BPM-C178S,K52N,G56C on different DNA promoters. The CCpmelR DNA is 186 bp containing the promoter region from -90 to +96; the CC+20pmelR DNA is 206 bp containing the promoter region from -110 to +96; the gal DNA is 209 bp containing the promoter region from -170 to +39; the lac DNA is 203 bp containing the promoter region from -140 to +63; The indicated DNA fragments were produced by PCR.



In order to determine the specificity of the cross-linking of CRP 52-loop with , another CRP mutant C178S,T158C was made with a cysteine substitution at the 158-position in the surface-exposed loop 156-162 present at the C-terminal domain of CRP. This mutant CRP retained about 60-70% of the stimulating activity on both gal and lac promoters in vivo compared to the wild type (data not shown). The same UV cross-linking reaction was carried out, and the results are shown in Fig. 4. C178S,T158C was found to cross-link with the alpha subunit of RNA polymerase consistent with previously published results (Chen et al., 1994), while no cross-linking was detected between this mutant and the subunit. Their results have shown that the 156-loop is close to the RNA polymerase alpha subunit on the lac promoter. Our results indicated that this loop can cross-link with alpha not only on lac but also on gal promoter and this cross-linking is strictly DNA-dependent.


Figure 4: Western blotting experiment showing the photo-cross-linking results for BPM-C178S,T158C on gal and lac DNA promoters. The gal and lac DNA promoters were produced by PCR as before.



To strengthen the conclusion that the cross-linking of CRP with relies on the functional preinitiation complex, substrate nucleotides (ATP, CTP, UTP, and OMeGTP) were incubated with the (BPM-(C178S,K52N,G56C))bulletgal PbulletRNA polymerase ternary complex mixture before UV irradiation. After a 5-min incubation at 37 °C, a 10-nucleotide-long transcript (terminated by the presence of OMeGTP) was allowed to form and then treated by UV irradiation as usual. The results indicated that following initiation of transcription and the clearance of the gal promoter, the cross-linked CRP- product was not produced to a significant extent (Fig. 5).


Figure 5: Western blotting experiment showing the photo-cross-linking results for BPM-C178S,K52N,G56C with RNA polymerase holoenzyme before or after the clearance of the gal DNA promoter. The reaction conditions given under ``Materials and Methods'' under Photo-cross-linking were used. A nucleotide substrate mixture (final concentration of 0.4 mM each ATP, CTP, UTP and 0.08 mM OMeGTP) was added to the CRPbulletgal PbulletRNA polymerase ternary complex followed by incubation at 37 °C for 5 min before UV irradiation.




DISCUSSION

Compelling evidence indicates that direct contact between RNA polymerase and CRP is required for transcription activation. In the case of Class I promoters such as lac P1 contact involves CRP loop-161 and the C-terminal region of the alpha subunit of RNA polymerase. In Class I promoters, the DNA site for CRP is centered at position -61.5 or further upstream (Ishihama, 1988). The involvement of the alpha subunit in activation is not limited to CRP but also includes a number of other transcription factors which bind to Class I sites (Ishihama, 1993).

Class II promoters such as gal P1 (CRP binding site centered at position -41.5) are affected to varying extents by CRP mutants which result in loss of transcription activation from Class I promoters. West et al.(1993) showed that amino acid substitution in the 159-contact loop (H159L,T158A) reduced CRP-dependent activation according to the Class II promoter used. Earlier work indicated that mutation in the loop involving amino acid 52 could reactivate the CRP mutation at position 159 (Bell et al., 1990). It was suggested by West et al.(1993) that mutation in the 52-loop could ``improve or create a second ``patch'' on CRP'' which may contact RNA polymerase.

We have provided direct evidence for contact between the 52-loop and the subunit of RNA polymerase. Cross-linking is specific for the subunit and requires the cAMP-bound conformer of CRP. Little cross-linking occurs in the absence of DNA. The mutants prepared for the present study show activity comparable to wild type CRP for lac expression. Mutant CRP C178S,K52N,G56C supports a 3-fold increased expression from the gal-like promoter, CCpmelR (Fig. 1). Replacing lysine 52 with asparagine (Bell et al., 1990) or glutamic acid (West et al., 1993) had previously been shown to stimulate expression from CCpmelR. Cross-linking carried out in the presence of promoter DNA shows the discrimination between lac-like and gal-like promoters.

Transcription factors can interact with RNA polymerase both in the presence and absence of promoter DNA (Ishihama, 1988). The contact site of CRP with RNA polymerase on Class I promoters has been located on the 159-loop of CRP and the C terminus of the alpha subunit of RNA polymerase. While the C terminus of alpha is indispensable for factor activation on Class I promoters, it does not appear to be essential for Class II promoter activation. On Class II promoters like gal P1, where the CRP binding site is located at the -41.5-position, it appears that protein-protein contact also plays an important role in formation of the CRPbulletRNA polymerasebulletDNA complex essential for transcription initiation. Kumar et al.(1994) demonstrated that deletions of the 4.2 conserved region of responsible for -35 recognition could still effectively transcribe gal P1 in the presence of cAMP-CRP. They also proposed that the CRP contact site for gal P1 activation could be located between amino acids 530 and 539 on . Our cross-linking results are consistent with their results in demonstrating that CRP physically contacts . Whether the region comprising amino acids 530-539 actually is the site for contact with CRP remains to be determined.

In order to demonstrate that the formation of a functional preinitiation complex on the gal promoter is necessary for the cross-linking between the CRP 52-loop and subunit of RNA polymerase, photo-cross-linking assays were carried out after the initiation of transcription. The ATP, CTP, UTP, and OMeGTP mixture was added to the preinitiation complex as substrates for transcription. Since the initial 10-ribonucleotide sequence of the gal P1 transcript is AUACCAUAAG . . . , this would enable transcription to initiate but pause after incorporation of the first 10 ribonucleotides because of the addition of the chain-terminating OMeGMP at the 10th position. Following photoirradiation, cross-linking between CRP and could hardly be detected after the initiation of transcription. This result clearly demonstrated that CRP- cross-linking relies on the formation of a functional transcription complex.

The surface-exposed loop of CRP spanning amino acids 156-162 has been shown to be in close contact with the C terminus of the alpha subunit of RNA polymerase on the lac promoter (Chen et al., 1994). To further establish the specificity of the cross-linking of our N-terminal CRP 52-loop mutations with the subunit, a cysteine substitution at the 158 position was made (C178S,T158C). This mutant was used for cross-linking reactions using the same conditions as for the 52-loop substitutions. The cross-linking results on the lac promoter were the same as those reported by Chen et al.(1994); C178S,T158C cross-linked with the alpha subunit of RNA polymerase on the lac promoter, while no cross-linking with beta, beta`, or was detected. This cross-linking was also cAMP- and DNA-dependent. Surprisingly, C178S,T158C also showed a similar efficiency for cross-linking with the alpha subunit on gal as well as lac promoters. Recently published evidence also indicated that CRP contacts the alpha subunit of RNA polymerase on the gal P1 promoter as demonstrated by DNA footprinting (Attey et al. 1994). They suggested that alpha binds at the upstream end of both the binary RNA polymerasebulletgal P1 and ternary RNA polymerasebulletCRPbulletgal P1 complexes. In the ternary complex, alpha appears to make direct contact with activating region 1 (which includes the 156-162-loop) in CRP. Taking this into consideration, our results showing cross-linking of C178S,T158C with alpha on both lac and gal promoters not only clarified the specificity of cross-linking (52-loop with and 158-loop with alpha), but also provided direct evidence that the activating region 1 of CRP makes direct contact with alpha on lac as well as on gal promoters.

No direct evidence is available indicating that the 52-loop of CRP is essential for stimulating transcription of gal P1. It remains possible that the contact between the CRP 52-loop and is gratuitous and not directly involved in transcription activation. It is also possible that more than one site on CRP may contact one or more RNA polymerase subunits for overall stimulating activity. So far, at least two of the RNA polymerase contact sites on CRP have been identified. West et al.(1993) suggest that neither the 52-loop nor the 159-loop is essential for activating transcription on Class II promoters, another activating region, perhaps located around glutamic acid 96, might also be involved in transcription stimulation.


FOOTNOTES

*
This work was supported by Research Grant GM22619 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Skirball Institute, New York University Medical Center, New York, NY 10016.

To whom correspondence and reprint requests should be addressed. Tel.: 212-772-5029; Fax: 212-772-5227.

(^1)
The abbreviations used are: CRP, cAMP receptor protein; BPM, benzophenone-4-maleimide; bp, base pair(s); PCR, polymerase chain reaction.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.