Identification of the functional subunit of a dimeric transcription activator protein by use of oriented heterodimers

Cell, Vol. 73, 375-379,

April 23, 1993, Copyright

0 1993 by Cell Press

Identification of the Functional Subunit of a Dimeric Transcription Activator Protein by Use of Oriented Heterodimers Yuhong Zhou,’ Steve Busby,tand Richard H. Ebright’ “Department of Chemistry and Waksman Institute Rutgers University New Brunswick, New Jersey 08855 tSchool of Biochemistry University of Birmingham Birmingham 815 2Tf England

Summary We have constructed heterodimers consisting of two subunits: one CAP subunit that has a nonfunctional activating region but wild-type DNA binding specificity, and one CAP subunit that has a functional activating region but non-wild-type DNA binding specificity. We have oriented the heterodimers on lac promoter DNA by use of promoter derivatives that have DNA sites for CAP consisting of one wild-type half site and one non-wild-type half site, and we have analyzed the abilities of the oriented heterodimers to activate transcription. Our results indicate that transcription activation requires the activating region of only one subunit of CAP: the promoter-proximal subunit. The oriented heterodimers method of this report should be generalizable to other dlmeric transcription activator proteins. Introduction The Escherichia coli catabolite gene activator protein (CAP; also referred to as the CAMP receptor protein, CRP) is a well-characterized transcription activator protein (reviewed by Pastan and Adhya, 1978; de Crombrugghe et al., 1984; Reznikoff, 1992). CAP activates transcription at a large set of promoters, the best characterized of which is the lac promoter. CAP functions by binding to a 22 bp P-fold-symmetric DNA site located in or upstream of each CAP-dependent promoter (Berg and von Hippel, 1988; Ebright et al., 1989; Gunasekeraet al., 1992). The three-dimensional structure of CAP has been determined to 2.5 A resolution by X-ray diffraction analysis (Weber and Steitz, 1987) and the three-dimensional structure of the CAP-DNA complex has been determined to 3.0 A resolution by X-ray diffraction analysis (Schultz et al., 1991). CAP is a dimer of two identical subunits, each of which is 209 amino acids in length. The CAP-DNA complex is 2-fold symmetric: one subunit of CAP interacts with one half of the DNA site; the other subunit of CAP interacts in a 2-fold-symmetry-related fashion with the other half of the DNA site. The majority of CAP-DNA interactions are mediated by the helix-turnhelix DNA-binding motif present in each subunit of CAP (reviews of helix-turn-helix motif by Brennan, 1991,1992). Two contacts between amino acids of the helix-turn-helix

motif of CAP and base pairs of the DNA half site have been demonstrated experimentally (Ebright et al., 1984, 1987; Zhang and Ebright, 1990); i.e., Arg-180 of CAP has been shown to contact base pair 5 of the DNA half site, and Glu-181 of CAP has been shown to contact base pair 7 of the DNA half site. Recent work has established that amino acids 156 to 162 of CAP constitute an activating region essential for transcription activation at the lac promoter but not essential for DNA binding or DNA bending (Bell et al., 1990; Williams et al., 1991; Eschenlauer and Reznikoff, 1991; Zhou et al., 1993). It has been proposed that transcription activation at the lac promoter requires a direct proteinprotein contact between the activating region of CAP and a molecule of RNA polymerase bound adjacent to CAP on the promoter DNA (Zhou et al., 1993; Heyduk et al., submitted). Because CAP is a dimer of two identical subunits, CAP hastwoactivating regions, one in each subunit. This raises the question: Does transcription activation at the lac promoter require the activating region of the promoterproximal subunit of the CAP dimer, the activating region of the promoter-distal subunit of the CAP dimer, or the activating regions of both subunits of the CAP dimer? To answer this question, we used a novel approach, which we term oriented heterodimers. Results Oriented Heterodimers: Approach Our approach had three steps. In step 1, we constructed heterodimers consisting of one CAP subunit that has a nonfunctional activating region but has wild-type DNA binding specificity, and one CAP subunit that has a functional activating region but has non-wild-type DNA binding specificity. In step 2, we oriented the resulting heterodimers on lac promoter DNA by use of lac promoter derivatives that have DNA sites for CAP consisting of one wild-type DNA half site and one non-wild-type DNA half site. In step 3, we assessed the abilities of the oriented heterodimers to activate transcription. We constructed heterodimers consisting of one [Ala158]CAP subunit and one [Val-181]CAP subunit. [Ala158]CAP has a nonfunctional activating region but has wild-type DNA binding specificity (Table 1; Zhou et al., 1993; Heyduket al., submitted; Y. Z. and R. H. E., unpublished data). [Ala-158]CAP, like wild-type CAP, exhibits strong specificity for G:C at position 7 of the DNA half site. [Ala-158]CAP is not able to bind with high affinity to DNA half sites containing A:T, C:G, or T:A at position 7. [Val181]CAP has a functional activating region but has nonwild-type DNA binding specificity (Table 1; Ebright et al., 1984, 1987). [Val-18l]CAP has an amino acid substitution at the amino acid of CAP that makes direct, specificitydetermining contact with the base pair at position 7 of the DNA half site (Ebright et al., 1984, 1987; Schultz et al., 1991). As a result, [Val-181]CAP exhibits essentially no

Cell 376

Table 1. CAP Subunits Subunit

Used

Activating

in the Analysis Region

DNA Half Site Recognized”

[Ala-156]CAP

Nonfunc0onal

1 A T

[Val-161]CAP

Functional

1 A T

2 A T

3 A T

4 T A

5 G C

6 T A

7 G c

6 A T

9 T A

10 C G

11 T A4

2 A T

3 A T

4 T A

5 G C

6 T A

7 N i

6 A T

9 T A

IO C G

11 T A4

’ Positions within the DNA half site are numbered as by de Crombrugghe et al. (1964), Ebright et al. (1964, 1967, 1969), Zhang Zhang 81 al. (1991, 1992), and Gunasekera et al. (1992). A different numbering convention is used by Schultz et al. (1991).

specificity at position 7 of the DNA half site. [Val-181JCAP binds with equal, high affinities to DNA half sites containing G:C, A:T, C:G, and T:A at position 7. To orient the [Ala-l 58]CAP-[Val-181]CAP heterodimers on lac promoter DNA, we used two derivatives of the lac promoter: lacL29 and lacL8 (Scaife and Beckwith, 1988; lppen et al., 1968; Dickson et al., 1977; Ebright et al., 1984). In each of these derivatives, 1 of the 2 DNA half sites of the DNA site for CAP is replaced by a DNA half site that has A:Tat position 7. In lacL29, the promoter-proximal DNA half site is non-wild type; in lacL8, the promoter-distal DNA half site is non-wild type. We expect that the [Ala-l 58]CAP-[Val-181JCAP heterodimer binds to lacL29 in an asymmetric, oriented fashion, since the [Ala-158]CAP subunit is able to bind with high affinity to the wild-type DNA half site but is not able to bind with high affinity to the non-wild-type DNA half site (Figure 1A). The [Ala-158]CAP-[Val-181]CAP heterodimer has onlyonefunctional activating region. At lacL29, the heterodimer is oriented such that the functional activating region is in the promoter-proximal subunit. Analogously, we expect that the [Ala-158]CAP-[Val-181JCAP heterodimer binds to lacL8 in an asymmetric, oriented fashion (Figure 1B). At lacL8, the heterodimer is oriented such that the functional activating region is in the promoter-distal subunit. Analysis of transcription activation by [Ala-l 58]CAP[Val-181JCAP heterodimers at lacL29 and lacL8 permits unambiguous determination of whether transcription activation at the lac promoter requires the activating region of the promoter-proximal subunit of the CAP dimer, the activating region of the promoter-distal subunit of the CAP dimer, or the activating regions of both subunits of the CAP dimer. There are three possible models. First, transcription activation might require the activating region of only one subunit of the CAP dimer: the promoter-proximal subunit. In this case, the heterodimerswould be functional in transcription activation at lacL29, but not at lacL8. Second, transcription activation might require the activating region of only one subunit of the CAP dimer: the promoterdistal subunit. In this case, the heterodimers would be functional in transcription activation at lacL8, but not at lacL29. Third, transcription activation might require the activating region of both subunits of the CAP dimer. In this case, the heterodimerswould be functional in transcription activation neither at lacL29 nor at lacL8. In control experiments, we constructed and analyzed

and Ebright

(1990),

[Val-181]CAP-[Val-18l]CAP homodimers. Weexpect that [Val-181]CAP-[Val-181JCAP homodimers bind to lacL29 and lacL8 in an unoriented fashion, since the [Val181]CAP subunit is able to bind with equal, high affinities to the wild-type DNA half site and the non-wild-type DNA half site (Figures 1C and 1D). The [Val-181]CAP-[Val181]CAP homodimer has two functional activating regions, one in each subunit. By any 1 of the 3 possible models described in the preceding paragraph, the [Val181]CAP-[Val-18l]CAP homodimer would be functional in transcription activation both at lacL29 and at lacL8. Oriented Heterodimers: Method CAP subunits exchange freely upon mixing (Brown and Crothers, 1989). To construct [Ala-158]CAP-[Val-1811 CAP heterodimers, we mixed a defined ratio of [Ala-1581 CAP subunits with [Val-181JCAP subunits. We used a large molar excess of [Ala-158]CAP subunits over [Val-181)CAP subunits (i.e., a lo-fold to 40-fold molar excess). Under such conditions, only two dimeric species are present in appreciable quantities: [Ala-158]CAP(Ala-158]CAP homodimers, which bind poorly to lacL29 and lacL8 and, therefore, do not affect the analysis; and [Ala-158]CAP-[Val-181JCAP heterodimers, thedesired dimerit species. We have performed experiments both in vivo and in vitro. To perform experiments in vivo, we constructed strains that coexpress the gene encoding [Ala-158]CAP at a gene dosage of -40 (expression from a high copy number plasmid) and the gene encoding [Val-181JCAP at a gene dosage of 1 (expression from the chromosome). In such strains, we analyzed transcription activation at lacL29 and lacL8 by measurement of differential rates of P-galactosidase synthesis (see Zhang et al., 1992; Zhou et al., 1993). In control experiments, we constructed strains that express only the gene encoding [Val-181]CAP (gene dosage of - 40; expression both from a high copy number plasmid and from the chromosome) and analyzed transcription activation at lacL29 and lacL8 by measurement of differential rates of P-galactosidase synthesis. To perform experiments in vitro, we preincubated a 1O-fold molar excess of [Ala-l 58]CAP with [Val-181]CAP under conditions that result in complete subunit exchange (500 nM [Ala-158]CAP, 50 nM [Val-181JCAP at 37OC for 24 hr; see Brown and Crothers, 1989). We then performed abortive initiation in vitro transcription experiments (Malan et al., 1984; Zhang et al., 1992; Zhou et al., 1993) using

Functional 377

Subunit

of a Dimeric

Transcription

Activator

L29

LB

C

D VlBi ml-

V181

Vi81 V?M

m

L29

Figure

1. Oriented

L8

Heterodimers

The figure illustrates the expected orientations of dimers at the lacL29 and lacL8 start point of the promoter, and the black bars indicate the -35 and -10 sites of (A) The [Ala-158]CAP-[Val-181]CAP heterodimer binds to lacL29 in an asymmetric, and it is in the promoter-proximal subunit. (B) The [Ala-156]CAP-[Val-lEll]CAP heterodimer binds to /a&B in an asymmetric, and it is in the promoter-distal subunit. (C and D) [Val-lEl]CAP-[Val-161JCAP homodimers bind to lacL29 and lacL8 in an one in each subunit

lacL29 or lacL8 as template. preincubated [Val-181]CAP tions (550 nM [Val-181]CAP performed abortive initiation ments using lacL29 or lacL8

In control experiments, we alone under identical condiat 37% for 24 hr) and then in vitro transcription experias template.

Oriented Heterodimers: Transcription Activation In Vivo and In Vitro The results of the experiments performed in vivo are presented in Table 2. The [Ala-l 58]CAP-[Val-181]CAP heterodimer was functional in transcription activation at lacL29, but was not functional in transcription activation at lacL8. The extent of transcription activation was 1 O-fold higher at lacL29 than at lacL8. In contrast, the [Val181)CAP-[Val-181]CAP homodimer was functional in transcription activation both at lacL29 and at lacL8. Within experimental error, the extent of transcription activation was equal at lacL29 and lacL8. These results indicate that transcription activation at the lac promoter requires the activating region of only one subunit of the CAP dimer: the promoter-proximal subunit. The results of the experiments performed in vitro are presented in Table 3. The results are in excellent agreement with the results of the experiments performed in vivo. Thus, the [Ala-158]CAP-[Val-181]CAP heterodimer was functional in transcription activation at lacL29, but was not functional in transcription activation at lacL8. The extent of transcription activation was 15fold higher at lacL29

Table 2. Transcription In Vivo Data

Activation

by Oriented 6-Galactosidase

Binding-Competent

Dimer

[Ala-158]CAP-[Val-1 ElICAP [Val-18l]CAP-[Val-18ljCAP a Units as by Miller (1972).

Heterodimers:

promoters. In each panel, the arrow indicates the transcription the promoter. oriented fashion. There is only one functional activating region, oriented

fashion.

unoriented

There

fashion.

There are two functional

lacL8

lacL29AacL8

1000 1100

100 850

lo

Table 3. Transcription In Vitro Data

activating

Activation

by Oriented

region, regions:

Heterodimers:

pmol of UMP Incorporated nmol of Template per min Binding-Competent

1.3

activating

than at lacL8. In contrast, the [Val-18l]CAP-[Val-181]CAP homodimer was functional in transcription activation both at lacL29 and at lacL8. Within experimental error, the extent of transcription activation was equal at lacL29 and lacL8. Again, these results indicate that transcription activation at the lac promoter requires the activating region of only one subunit of the CAP dimer: the promoter-proximal subunit. [Leu-159]CAP, like [Ala-l 58]CAP, has a nonfunctional activating region but has wild-type DNA binding specificity (Bell et al., 1990; Zhou et al., 1993; Y. Z. and R. H. E., unpublished data). We have performed analogous experiments with [Leu-159]CAP-[Val-181]CAP heterodimers. The experiments with [Leu-159]CAP-[Val-181jCAP heterodimers yielded results equivalent to those with [Ala-1581 CAP-[Val-181)CAP heterodimers. Thus, [Leu-159]CAP[Val-181]CAP heterodimers were functional in transcription activation at lacL29, but were not functional in transcription activation at lacL8; i.e., the extent of transcription activation was 14-fold higher at lacL29 than at lacL8. The results with [Leu-159]CAP-[Val-181JCAP heterodimers rule out the possibility that the results with [Ala-158]CAP[Val-1811 heterodimers are artifacts due to specific, unrepresentative effects of the Thr-158-Ala substitution. Wild-type CAP, of course, has a functional activating region and wild-type DNA binding specificity. We expect that aCAP-[Val-181]CAP heterodimer bindstolacL29and to lacL8 in an asymmetric, oriented fashion (like the [Ala-

Synthesis’

lacL29

is only one functional

Dimer

[Ala-156]CAP-[Val-lBl]CAP [Val-16l]CAP-[Val-16ljCAP

per

lacL29

lacL8

lacL29AacL8

20 26

1.3 39

7.67

15

Cell 378

Table 4. Bacterial

Strains

Strain

Genotype

Source

XE832.1 XE832.11158A XE832.111181V

lacL8 Aga1765 thi crpl8lV lacL8 Aga1165 thi crpl8lV lacL8

Ebright et al., 1984 This study This study

XE932.1 XE932.11158A XE932.11181V

lacL29 lacL29 lacL29

dga1765

thi crp787V

pYZ158A pPC181V

Agal thi crpl8lV Agall 65 thi crpl81 V pYZ158A Aga1765 thi crpl8lV pPC181V

158]CAP-[Val-181]CAP heterodimer), but has functional activating regions in both subunits (like the [Val-181]CAP[Val-181]CAP homodimer). We have performed analogous experiments with CAP-[Val-181]CAP heterodimers. The experiments with CAP-[Val-181)CAP heterodimers yielded results equivalent to those with [Val-181]CAP[Val-181]CAP homodimers. Thus, CAP-[Val-181]CAP heterodimers were functional in transcription activation both at lacL29 and at lacL8. This rules out the possibility that our results with [Ala-158]CAP-[Val-181]CAP heterodimers are artifacts of heterodimer formation per se or of heterodimer orientation per se. Discussion The results in this report establish that transcription activation at the lac promoter requires the activating region of only one subunit of the CAP dimer: the promoter-proximal subunit. The results in this report support the proposal that transcription activation at the lac promoter requires a direct protein-protein contact between the activating region of CAP and a molecule of RNA polymerase bound adjacent to CAP on the promoter DNA (Zhou et al., 1993; Heyduk et al., submitted). By any simple model for the structure of a CAP-RNA polymerase-lac promoter ternary complex, a direct protein-protein contact between CAP and RNA polymerase would be expected to involve the activating region of the promoter-proximal subunit of CAP (Zhou et al., 1993). The oriented heterodimers approach of this report should be generalizable to any dimeric transcription activator protein for which both mutants specifically defective in transcription activation and mutants with relaxed or altered DNA binding specificity are available. Experimental

Procedures

Strains, Plasmids, Media, A list of E. coli K-12 strains

and Microbiological Techniques used in this work is presented in Table

4,

Ebright et al., 1984 This study This study

and a list of plasmids used in this work is presented in Table 5. Standard media were prepared and standard genetic manipulations were performed as described by Miller (1972). CAP Derivatives [Ala-158]CAP and [Val-lBl]CAP were purified by CAMP affinity chromatography followed by gel filtration chromatography as described by Zhang et al. (1991).

Measurement of Transcription Activation in Vivo Experimentswith [Ala-158]CAP-[Vai-181JCAP heterodimerswereperformed using strains XE932.11158A and XE832.11158A (Table 4). Experiments with [Val-lBl]CAP-[Val-181jCAP homodimers were performed using strains XE932.11181V and XE832.11181V (Table 4). Differential rates of !3-galactosidase synthesis were determined as described by Miller (1972), except that cultures were grown in LB medium containing 100 pglmi ampicillin and 1 mM isopropyl+-D-thiogalactopy ranoside. Data reported are the means of four independent determinations and have been corrected for CAP-independent B-gaiactosidase synthesis (86 U for lacL29; 81 U for lacL8).

Measurement of Transcription Activation in Vitro Subunit exchange reaction mixtures (190 ~1) contained 500 nM [Aia158jCAP subunit and 50 nM [Vai-181jCAP subunit (or 0 nM [Ala158)CAP subunit and 550 nM [Vai-181jCAP subunit), 40 mM Tris-HCI (pH 8.0), 100 mM KCI, 10 mM MgClz, 1 mM dithiothreitoi, and 5% glycerol. Reactions proceeded for 24 hr at 37OC. Abortive initiation in vitro transcription experiments (Maian et al., 1984; Zhang et al., 1992; Zhou et al., 1993) were performed using as templates a 203 bp EcoRI-EcoRI DNA fragment of plasmid pBR-203 lacPL29 and a203 bp EcoRI-EcoRI DNAfragmentof piasmid pBR-203. lacPL8. Reaction mixtures (25 ~1) contained 100 nM [Ala-158JCAP subunit and IO nM [Vai-181jCAP subunit (or 0 nM [Ala-158]CAP subunit and 110 nM [Vai-1 8lJCAP subunit), 0.5 nM DNA fragment, 40 nM E. coli RNA polymerase hoioenzyme (purified as described by Hager et al. [1990]), 0.5 mM ApA (ICN Biomedicais, inc.), 50 nM [aPP]UTP (30 Bq/fmol), 40 mM Tris-HCI (pH 8.0), 100 mM KCI, 10 mM MgCi2, 1 mM dithiothreitoi, 5% glycerol, and 0.2 mM CAMP. Reaction components except ApA and [a-=P]UTP were preequilibrated for IO min at 37QC. Reactions were initiated by addition of ApA and [aJ2P]UTP and were allowed to proceed for 15 min at 37OC. Reactions were terminated by addition of 5 pi of 0.5 M EDTA. The reaction product p2PlApApUpU was resolved by paper chromatography in water:saturated ammonium sulfate:isopropanoi (18:80:2, v/v; R, = 0.05) and was quantified by Cer. enkov counting. Data reported are meansof four independent determi. nations and have been corrected for CAP-independent incorporation.

Table 5. Plasmids Plasmid

Relevant

pYZ158A pPC181V pBR.203.lacPL29 pBR-203.lacPL8

ApR; ApR; ApR; Ap?

Characteristics

ori-pBR322; ori-pBR322; ori-pBR322; ori-pBR322;

ori-fl; crp758A crpl81V 203 bp EcoRI-EcoRI 203 bp EcoRI-EcoRl

Source

DNA fragment DNA fragment

containing containing

lacL29 promoter lacL8 promoter

Zhou et al., 1993 Ebright et al., 1987 A. Kolb and H. But A. Kolb and H. But

Functional 379

Subunit

of a Dimeric

Transcription

Activator

Acknowledgments We thank Drs. A. Kolb and H. But for plasmids pBR-203-lacPL29 and pBR-203-lacPL8. This work was supported by National Institutes of Health grant GM41376 to Ft. H. E., an EPA Cephalosporin Research Fellowship to S. B., and a Busch Predoctoral Fellowship to Y. 2. Received

January

13, 1993; revised

February

Weber, I., and Steitz, T. (1987). Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 A resolution. J. Mol. Biol. 798, 31 l-326. Williams, R., Bell, A., Sims, G., and Busby, S. (1991). The role of two surface exposed loops in transcription activation by the Escherichia co/i CRP and FNR proteins. Nucl. Acids Res. 79, 6705-6712. Zhang, X., and Ebright, R. (1990). Identification of a contact between arginine-180 of the catabolite gene activator protein (CAP) and base pair 5 of the DNA site in the CAP-DNA complex. Proc. Natl. Acad. Sci. USA 87, 4717-4721.

5, 1993.

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Berg, O., and von Hippel, P. (1986). Selection of DNA binding sites by regulatory proteins. The binding specificity of cyclic AMP receptor protein to recognition sites. J. Mol. Biol. 200, 709-723.

Zhang, X., Zhou, Y., Ebright, Y., and Ebright, R. (1992). Catabolite gene activator protein (CAP) is not an “acidic activating region” transcription activator protein: negatively charged amino acids of CAP that are solvent-accessible in the CAP-DNA complex play no role in transcription activation at the lac promoter. J. Biol. Chem. 267,81368139.

Brennan, R. (1991). Interactionsof the helix-turn-helix Curr. Opin. Struct. Biol. 7, 80-88. Brennan, R. (1992). DNArecognition Opin. Struct. Biol. 2, 100-108.

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Ebright, R., Kolb, A., But, H., Kunkel, T., Krakow, J., and Beckwith, J. (1987). Role of glutamic acid-181 in DNA-sequence recognition by the catabolite gene activator protein (CAP) of Escherichie co/i: altered DNA-sequence-recognition properties of [Vall81]CAP and [LeulBl]CAP. Proc. Natl. Acad. Sci. USA 84, 6083-6067. Ebright, R., Ebright, Y., and Gunasekera, A. (1969). Consensus DNA site for the Escherichia co/i catabolite gene activator protein (CAP): CAP exhibits a 450-fold higher affinity for the consensus DNA site than for the E. co/i lac DNA site. Nucl. Acids Res. 77, 10295-10305. Eschenlauer, A., and Reznikoff, gene activator protein mutants eron transcription. J. Bacterial.

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(Cold Spring

Har-

5’-monophosphate protein

activation

of

Scaife, J., and Beckwith, J. (1966). Mutational alteration of the maximal level of lac operon expression. Cold Spring Harbor Symp. Quant. Biol. 37,403-406. Schultz, S., Shields, CAP-DNA complex: 1001-1007.

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Zhou, Y., Zhang, X., and Ebright. R. (1993). Identification of theactivating region of CAP: isolation and characterization of mutants of CAP specifically defective in transcription activation. Proc. Natl. Acad. Sci. USA, in press.