Publications

1984
Ebright, RH, Cossart P, Gicquel-Sanzey B, Beckwith J.  1984.  Molecular basis of DNA sequence recognition by the catabolite gene activator protein: detailed inferences from three mutations that alter DNA sequence specificity.. Proceedings of the National Academy of Sciences of the United States of America. 81(23):7274-8. Abstract
Previously, we reported that substitution of Glu-181 of the catabolite gene activator protein (CAP) by lysine, leucine, or valine results in a protein that has specificity for A X T base pairs at positions 7 and 16 of the DNA recognition site, rather than G X C base pairs as is the case with the wild-type CAP. In this paper, we deduce from these genetic data both (i) the specific chemical interactions by which amino acid side chains at position 181 interact with base pairs 7 and 16 and (ii) the precise alignment between the structures of the CAP and DNA in the intermolecular CAP-DNA complex. Our analysis supports the idea that the two symmetry-related F alpha-helices of the CAP dimer interact with successive major grooves of right-handed B-type DNA [Pabo, C. & Lewis, M. (1982) Nature (London) 298, 443-447; and Steitz, T., Weber, I. & Matthew, J. (1983) Cold Spring Harbor Symp. Quant. Biol. 47, 419-426].
Ebright, RH, Cossart P, Gicquel-Sanzey B, Beckwith J.  1984.  Mutations that alter the DNA sequence specificity of the catabolite gene activator protein of E. coli.. Nature. 311(5983):232-5. Abstract
Three mutations that alter the DNA sequence specificity of the catabolite gene activator protein (CAP) from AA-TGTGA--T---TCA-ATW to AA-TGTAA--T---TCA-ATW have been isolated. All three mutations affect the same amino acid of CAP, glutamic acid 181. We propose that it is this amino acid of CAP that makes contacts with base pairs 7 and 16 of the symmetrical recognition site.
1985
Ebright, RH, Beckwith J.  1985.  The catabolite gene activator protein (CAP) is not required for indole-3-acetic acid to activate transcription of the araBAD operon of Escherichia coli K-12.. Molecular & general genetics : MGG. 201(1):51-5. Abstract
Kline et al. (1980) have reported that indole-3-acetic acid (IAA) and four other indole derivatives are able to substitute for cAMP in activating expression of the ara regulon of E. coli. We have examined this phenomenon in detail, utilizing fusions between the structural gene for beta-galactosidase and the promoters for the araBAD, araE, and araFG operons. We confirm that IAA potently stimulates transcription from the araBAD promoter. The effect is highly specific to araBAD, as IAA has no, or only slight, effects on the araE and araFG operons. However, contrary to the results of Kline et al., we find that the action of IAA does not require CAP. Thus, IAA fully stimulates the transcription of araBAD in a strain which bears a complete deletion of the crp gene.
Duax, WL, Griffin JF, Ebright R.  1985.  Structural basis for chemotherapeutic action of antiestrogens.. Progress in clinical and biological research. 172B:263-73.
Ebright, RH, Le Grice SF, Miller JP, Krakow JS.  1985.  Analogs of cyclic AMP that elicit the biochemically defined conformational change in catabolite gene activator protein (CAP) but do not stimulate binding to DNA.. Journal of molecular biology. 182(1):91-107. Abstract
We have measured the effects on catabolite gene activator protein (CAP) of 22 synthetic analogs of cAMP. Each analog was assayed to test three parameters: (1) binding to CAP; (2) induction of the conformational change in CAP; and (3) activation of transcription. Thus we have identified seven cAMP analogs that bind to CAP as well or better than does cAMP, cause the assayed conformational change in CAP, yet exhibit no ability to activate transcription. We designate these analogs class D. The conformational change elicited in CAP by the class D analogs was further investigated by: (1) sensitivity to the proteolytic enzymes chymotrypsin, Staphylococcus aureus V8 protease, subtilisin and trypsin; (2) formation of inter-subunit covalent crosslinks by 5,5'-dithiobis(2-nitrobenzoic acid); and (3) degree of labeling of cysteine by [3H]N-ethylmaleimide. These experiments failed to detect a conformational difference between the CAP-class D and CAP-cAMP complexes. Filter binding and nuclease protection experiments indicate that the class D analogs do not efficiently support the binding of CAP to DNA. From these results, we suggest that there exists a hitherto undetected event dependent on cAMP, and required for CAP to bind to DNA. We suggest that this event involves a change that takes place in proximity to the N6 atom of cAMP. Three possible interpretations are discussed.
Ebright, RH.  1985.  Use of "loss-of-contact" substitutions to identify residues involved in an amino acid-base pair contact: effect of substitution of Gln18 of lac repressor by Gly, Ser, and Leu.. Journal of biomolecular structure & dynamics. 3(2):281-97. Abstract
A procedure to identify which base pair of lac operator (lacO) a suspected contacting amino acid of Lac repressor (LacR) interacts with is presented. The procedure is to eliminate the ability of the amino acid under study to contact DNA, and then to determine at which base pair--if any--specificity is eliminated. To implement this procedure, four sets of Escherichia coli K-12 strains have been constructed. These strains permit: (i) the substitution of a selected amino acid of LacR by, respectively, Gly, Ser, Leu, or Gln, and (ii) the analysis of the specificity of the resulting substituted LacR with respect to base pairs 5, 6, 7, 8, 9, and 10 of lacO. This procedure has been applied to Gln18 of LacR. The preliminary data indicate that LacR (Gln18----Gly) is unable to distinguish between the O+ base pair G:C and the Oc base pair T:A at position 7 of lacO (KDOc/KDO+ = 0.93). In contrast, LacR(Gln18----Gly) discriminates O+ from Oc by a factor of 13 to 23 at each other position. The same qualitative pattern of results was obtained with LacR(Gln18----Ser) and LacR (Gln18----Leu). Therefore, I propose that Gln18 contacts base pair 7 of lacO. This proposal is consistent with the contact predicted in Ebright, R. in Protein Structure, Folding, and Design. D. Oxender ed., Alan R. Liss, New York (1985), in press.
1986
Ebright, RH.  1986.  Evidence for a contact between glutamine-18 of lac repressor and base pair 7 of lac operator.. Proceedings of the National Academy of Sciences of the United States of America. 83(2):303-7. Abstract
Glutamine-18 of the lac repressor (lacR) has been substituted by glycine, by serine, and by leucine. The specificities of wild-type lacR and of the three substituted lacR variants have been analyzed with respect to base pairs 5, 6, 7, 8, 9, and 10 of the lac operator (lacO). The data indicate that [Gly18]lacR, [Ser18]lacR, and [Leu18]lacR lose the ability to distinguish between the O+ base pair G . C and the Oc base pairs T . A and A . T at position 7 of lacO (KdOc/KdO+ approximately equal to 1). In contrast, the three substituted variants retain the ability to discriminate O+ from Oc at each other position, by factors of 9 to 37. Therefore, I propose that glutamine-18 contacts base pair 7 of lacO. These data suggest that the interaction between the helix-turn-helix motif and DNA may be very similar or identical in lacR and the catabolite gene activator protein.
Ebright, RH, Wong JR, Chen LB.  1986.  Binding of 2-hydroxybenzo(a)pyrene to estrogen receptors in rat cytosol.. Cancer research. 46(5):2349-51. Abstract
The potent carcinogen 2-hydroxybenzo(a)pyrene (2-OH-BP) competes for binding to the estrogen receptor in the cytosol of rat uterus and liver. The dissociation constant (K1) for this interaction is congruent to 2 X 10(-5) M. In contrast, 4-hydroxybenzo(a)pyrene does not bind to the estrogen receptor; 1-hydroxybenzo(a)pyrene, 5-hydroxybenzo(a)pyrene, 6-hydroxybenzo(a)pyrene, and 12-hydroxybenzo(a)pyrene bind less tightly than does 2-OH-BP. These five chemicals are not carcinogenic. We suggest that the estrogen receptor may mediate the carcinogenic effect of 2-OH-BP or of related chemicals. One possibility is that the receptor might convey 2-OH-BP to specific sites in DNA.
1987
Ebright, RH, Kolb A, Buc H, Kunkel TA, Krakow JS, Beckwith J.  1987.  Role of glutamic acid-181 in DNA-sequence recognition by the catabolite gene activator protein (CAP) of Escherichia coli: altered DNA-sequence-recognition properties of [Val181]CAP and [Leu181]CAP.. Proceedings of the National Academy of Sciences of the United States of America. 84(17):6083-7. Abstract
It has been proposed that Glu-181 of the catabolite gene activator protein (CAP) makes direct contact with certain base pairs of the specific DNA site. We have purified wild-type CAP and two substituted CAP variants, [Val181]CAP and [Leu181]CAP, and have assessed the DNA-sequence-recognition properties in vitro with respect to positions 5, 6, 7, 8, and 16 of the DNA site. The data indicate that [Val181]CAP and [Leu181]CAP fail to discriminate between the consensus DNA base pair and the three non-consensus-DNA base pairs at 2-fold-related positions 7 and 16 of the DNA site. In contrast, [Val181]CAP and [Leu181]CAP retain the ability to discriminate between different base pairs at positions 5 and 8 of the DNA site. We conclude that Glu-181 of CAP makes a direct contact with 2-fold-related positions 7 and 16 of the DNA site, as proposed previously based on in vivo results. We propose that upon replacement of Glu-181 by valine or leucine, this contact is eliminated and is replaced by no other functional contact. We estimate that the contact by Glu-181 with each position contributes -0.7 kcal/mol to the total CAP-DNA binding free energy.
1989
Ebright, RH, Ebright YW, Gunasekera A.  1989.  Consensus DNA site for the Escherichia coli catabolite gene activator protein (CAP): CAP exhibits a 450-fold higher affinity for the consensus DNA site than for the E. coli lac DNA site.. Nucleic acids research. 17(24):10295-305. Abstract
We have synthesized two 40 base pair DNA fragments; one fragment contains the consensus DNA site for CAP (fragment 'ICAP'); the other fragment contains the E. coli lac promoter DNA site for CAP (fragment 'LCAP'). We have investigated the binding of CAP to the two DNA fragments using the nitrocellulose filter binding assay. Under standard conditions [( NaCl] = 200 mM, pH = 7.3), CAP exhibits a 450-fold higher affinity for ICAP than for LCAP. The salt dependence of the binding equilibrium indicates that CAP makes eight ion pairs with ICAP, but only six ion pairs with LCAP. Approximately half of the difference in binding free energy for interaction of CAP with ICAP vs. LCAP is attributable to this difference in ion-pair formation. The pH dependence of the binding equilibrium indicates that the eight CAP-ICAP ion pairs and the six CAP-LCAP ion pairs do not involve His residues of CAP.
1990
Ebright, RH, Ebright YW, Pendergrast PS, Gunasekera A.  1990.  Conversion of a helix-turn-helix motif sequence-specific DNA binding protein into a site-specific DNA cleavage agent.. Proceedings of the National Academy of Sciences of the United States of America. 87(8):2882-6. Abstract
Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein [de Crombrugghe, B., Busby, S. & Buc, H. (1984) Science 224, 831-838; and Pabo, C. & Sauer, R. (1984) Annu. Rev. Biochem. 53, 293-321]. In this work, CAP has been converted into a site-specific DNA cleavage agent by incorporation of the chelator 1,10-phenanthroline at amino acid 10 of the helix-turn-helix motif. [(N-Acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP binds to a 22-base-pair DNA recognition site with Kobs = 1 x 10(8) M-1. In the presence of Cu(II) and reducing agent, [(N-acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP cleaves DNA at four adjacent nucleotides on each DNA strand within the DNA recognition site. The DNA cleavage reaction has been demonstrated using 40-base-pair and 7164-base-pair DNA substrates. The DNA cleavage reaction is not inhibited by dam methylation of the DNA substrate. Such semisynthetic site-specific DNA cleavage agents have potential applications in chromosome mapping, cloning, and sequencing.
Gunasekera, A, Ebright YW, Ebright RH.  1990.  DNA-sequence recognition by CAP: role of the adenine N6 atom of base pair 6 of the DNA site.. Nucleic acids research. 18(23):6853-6. Abstract
Two similar, but not identical, models have been proposed for the amino acid-base pair contacts in the CAP-DNA complex ('Model I,' Weber, I. and Steitz, T., Proc. Natl. Acad. Sci. USA, 81, 3973-3977, 1984; 'Model II,' Ebright, et al., Proc. Natl. Acad. Sci. USA, 81, 7274-7278, 1984). One difference between the two models involves Glu181 of CAP. Model I predicts that Glu181 of CAP makes two specificity determining contacts: one H-bond with the cytosine N4 atom of G:C at base pair 7 of the DNA half site, and one H-bond with the adenine N6 atom of T:A at base pair 6 of the DNA half site. In contrast, Model II predicts that Glu181 makes only one specificity determining contact: one H-bond with the cytosine N4 atom of G:C at base pair 7 of the DNA half site. In the present work, we show that replacement of T:A at base pair 6 of the DNA half site by T:N6-methyl-adenine has no, or almost no, effect on the binding of CAP. We conclude, contrary to Model I, that Glu181 of CAP makes no contact with the adenine N6 atom of base pair 6 of the DNA half site.
Zhang, XP, Ebright RH.  1990.  Substitution of 2 base pairs (1 base pair per DNA half-site) within the Escherichia coli lac promoter DNA site for catabolite gene activator protein places the lac promoter in the FNR regulon.. The Journal of biological chemistry. 265(21):12400-3. Abstract
The consensus DNA site for Escherichia coli catabolite gene activator protein (CAP) is 5'-AAATGTGATCTAGATCACATTT-3'. The proposed consensus DNA site for E. coli FNR is 5'-AAATTTGATATATATCAAATTT-3'. In this report, we show that substitution of 2 base pairs (1 base pair per DNA half-site) within the E. coli lac DNA site for CAP suffices to remove the lac promoter from the CAP regulon and to place the lac promoter in the FNR regulon. FNR stimulates transcription of the derivative of the lac promoter having G:C----T:A substitutions at base pair 5 each DNA half-site (13-fold stimulation). FNR does not stimulate transcription of the wild-type lac promoter, or of derivatives of the lac promoter having G:C----A:T or G:C----C:G substitutions at base pair 5 of each DNA half-site. Stimulation of transcription is strictly dependent on anaerobiosis. FNR-stimulated transcription initiates at the same base pair as does CAP-dependent transcription of the wild-type lac promoter.
Zhang, XP, Ebright RH.  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.. Proceedings of the National Academy of Sciences of the United States of America. 87(12):4717-21. Abstract
We have used site-directed mutagenesis to replace amino acid 1 of the recognition alpha-helix of the catabolite gene activator protein (CAP), Arg-180, with glycine and with alanine. Substitution of Arg-180 of CAP eliminated specificity between G.C, A.T, C.G, and T.A at base pair 5 of the DNA half-site. The effect was position-specific: substitution of Arg-180 did not eliminate specificity between G.C, A.T, C.G, and T.A at base pair 7 of the DNA half-site. We conclude, in agreement with the model for the structure of the CAP-DNA complex [Weber, I. & Steitz, T. (1984) Proc. Natl. Acad. Sci. USA 81, 3973-3977; and Ebright, R., Cossart, P., Gicquel-Sanzey, B. & Beckwith, J. (1984) Proc. Natl. Acad. Sci. USA 81, 7274-7278], that Arg-180 of CAP makes a specificity-determining contact with base pair 5 of the DNA half-site in the CAP-DNA complex. The identification of the contact by Arg-180 in this report, in conjunction with the identification of the contact by Glu-181 in a previous report [Ebright, R., Cossart, P., Gicquel-Sanzey, B. & Beckwith, J. (1984) Nature (London) 311, 232-235], provides information sufficient to define the orientation of the helix-turn-helix motif of CAP with respect to DNA in the CAP-DNA complex.
Ebright, RH, Gunasekera A, Zhang XP, Kunkel TA, Krakow JS.  1990.  Lysine 188 of the catabolite gene activator protein (CAP) plays no role in specificity at base pair 7 of the DNA half site.. Nucleic acids research. 18(6):1457-64. Abstract
Two similar, but not identical, models have been proposed for the amino acid-base pair contacts in the CAP-DNA complex ('model I,' Weber, I. and Steitz, T., Proc. Natl. Acad. Sci. USA, 81, 3973-3977, 1984; 'model II,' Ebright, et al., Proc. Natl. Acad. Sci. USA, 81, 7274-7278, 1984). The most important difference between the two models involves Lys188 of CAP. Model I predicts that Lys188 of CAP makes a specificity determining contact with base pair 7 of the DNA half site. In contrast, model II predicts that Lys188 makes no contact with base pair 7 of the DNA half site. In the present work, we have used site-directed mutagenesis to replace Lys188 of CAP by Asn, an amino acid unable to make the putative contact. We have assessed the specificities of the following proteins, both in vitro and in vivo: wild-type CAP, [Asn188]CAP, [Val181]CAP, and [Val181;Asn188]CAP. The results indicate that Lys188 makes no contribution to specificity at base pair 7 of the DNA half site. We propose, contrary to model I, that Lys188 makes no contact with base pair 7 of the DNA half site.
1991
Ebright, RH.  1991.  Identification of amino acid-base pair contacts by genetic methods.. Methods in enzymology. 208:620-40.
Zhang, XP, Gunasekera A, Ebright YW, Ebright RH.  1991.  Derivatives of CAP having no solvent-accessible cysteine residues, or having a unique solvent-accessible cysteine residue at amino acid 2 of the helix-turn-helix motif.. Journal of biomolecular structure & dynamics. 9(3):463-73. Abstract
The Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein. CAP contains a unique solvent-accessible cysteine residue at amino acid 10 of the helix-turn-helix motif. In published work, we have constructed a prototype semi-synthetic site-specific DNA cleavage agent from CAP by use of cysteine-specific chemical modification to incorporate a nucleolytic chelator-metal complex at amino acid 10 of the helix-turn-helix motif [Ebright, R., Ebright, Y., Pendergrast, P.S. and Gunasekera, A., Proc. Natl. Acad. Sci. USA 87, 2882-2886 (1990)]. Construction of second-generation semi-synthetic site-specific DNA cleavage agents from CAP requires the construction of derivatives of CAP having unique solvent-accessible cysteine residues at sites within CAP other than amino acid 10 of the helix-turn-helix motif. In the present work, we have constructed and characterized two derivatives of CAP having no solvent-accessible cysteine residues: [Ser178]CAP and [Leu178]CAP. In addition, in the present work, we have constructed and characterized one derivative of CAP having a unique solvent-accessible cysteine residue at amino acid 2 of the helix-turn-helix motif: [Cys170;Ser178]CAP.
Zhou, YH, Zhang XP, Ebright RH.  1991.  Random mutagenesis of gene-sized DNA molecules by use of PCR with Taq DNA polymerase.. Nucleic acids research. 19(21):6052.
Shin, JA, Ebright RH, Dervan PB.  1991.  Orientation of the Lac repressor DNA binding domain in complex with the left lac operator half site characterized by affinity cleaving.. Nucleic acids research. 19(19):5233-6. Abstract
Lac repressor (LacR) is a helix-turn-helix motif sequence-specific DNA binding protein. Based on proton NMR spectroscopic investigations, Kaptein and co-workers have proposed that the helix-turn-helix motif of LacR binds to DNA in an orientation opposite to that of the helix-turn-helix motifs of lambda repressor, lambda cro, 434 repressor, 434 cro, and CAP [Boelens, R., Scheek, R., van Boom, J. and Kaptein, R., J. Mol. Biol. 193, 1987, 213-216]. In the present work, we have determined the orientation of the helix-turn-helix motif of LacR in the LacR-DNA complex by the affinity cleaving method. The DNA cleaving moiety EDTA.Fe was attached to the N-terminus of a 56-residue synthetic protein corresponding to the DNA binding domain of LacR. We have formed the complex between the modified protein and the left DNA half site for LacR. The locations of the resulting DNA cleavage positions relative to the left DNA half site provide strong support for the proposal of Kaptein and co-workers.
1992
Zhang, X, Zhou Y, Ebright YW, Ebright RH.  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 lac.. The Journal of biological chemistry. 267(12):8136-9. Abstract
It has been suggested that the catabolite gene activator protein (CAP) uses an "acidic activating region" transcription activation mechanism and that Glu171 of CAP is the critical amino acid of the "acidic activating region" of CAP (Irwin, N., and Ptashne, M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8315-8319). In this paper, we show, contrary to the previously published report, that substitution of Glu171 of CAP fails to result in a specific defect in transcription activation at the lac promoter. Furthermore, in this paper, we show that substitution of each other negatively charged amino acid of CAP that is solvent-accessible in the CAP-DNA complex fails to result in a specific defect in transcription activation at the lac promoter. We conclude that CAP does not use an acidic activating region transcription activation mechanism in transcription activation at the lac promoter.
Dong, Q, Ebright RH.  1992.  DNA binding specificity and sequence of Xanthomonas campestris catabolite gene activator protein-like protein.. Journal of bacteriology. 174(16):5457-61. Abstract
The Xanthomonas campestris catabolite gene activator protein-like protein (CLP) can substitute for the Escherichia coli catabolite gene activator protein (CAP) in transcription activation at the lac promoter (V. de Crecy-Lagard, P. Glaser, P. Lejeune, O. Sismeiro, C. Barber, M. Daniels, and A. Danchin, J. Bacteriol. 172:5877-5883, 1990). We show that CLP has the same DNA binding specificity as CAP at positions 5, 6, and 7 of the DNA half site. In addition, we show that the amino acids at positions 1 and 2 of the recognition helix of CLP are identical to the amino acids at positions 1 and 2 of the recognition helix of CAP:i.e., Arg at position 1 and Glu at position 2.
Gunasekera, A, Ebright YW, Ebright RH.  1992.  DNA sequence determinants for binding of the Escherichia coli catabolite gene activator protein.. The Journal of biological chemistry. 267(21):14713-20. Abstract
The consensus DNA site for binding of the Escherichia coli catabolite gene activator protein (CAP) is 22 base pairs in length and is 2-fold symmetric: 5'-AAATGTGATCTAGATCACATTT-3'. Positions 4 to 8 of each half of the consensus DNA half-site are the most strongly conserved. In this report, we analyze the effects of substitution of DNA base pairs at positions 4 to 8, the effects of substitution of thymine by uracil and by 5-methylcytosine at positions 4, 6, and 8, and the effect of dam methylation of the 5'-GATC-3' sequence at positions 7 to 10. All DNA sites having substitutions of DNA base pairs at positions 4 to 8 exhibit lower affinities for CAP than does the consensus DNA site, consistent with the proposal that the consensus DNA site is the ideal DNA site for CAP. Specificity for T:A at position 4 appears to be determined solely by the thymine 5-methyl group. Specificity for T:A at position 6 and specificity for A:T at position 8 appear to be determined in part, but not solely, by the thymine 5-methyl group. dam methylation has little effect on CAP.DNA complex formation. The thermodynamically defined consensus DNA site spans 28 base pairs. All, or nearly all, DNA determinants required for maximal affinity for CAP and for maximal thermodynamically defined CAP.DNA ion pair formation are contained within a 28-base pair DNA fragment that has the 22-base pair consensus DNA site at its center. The quantitative data in this report provide base-line thermodynamic data required for detailed investigations of amino acid-base pair and amino acid-phosphate contacts in this protein-DNA complex.
Ebright, R, Dong Q, Messing J.  1992.  Corrected nucleotide sequence of M13mp18 gene III.. Gene. 114(1):81-3. Abstract
There are seven differences between the actual nucleotide (nt) sequence of bacteriophage M13mp18 gene III and the previously reported nt sequence (which had been compiled based on the nt sequence of wild-type bacteriophage M13 gene III).
Pendergrast, PS, Chen Y, Ebright YW, Ebright RH.  1992.  Determination of the orientation of a DNA binding motif in a protein-DNA complex by photocrosslinking.. Proceedings of the National Academy of Sciences of the United States of America. 89(21):10287-91. Abstract
We have developed a straightforward biochemical method to determine the orientation of the DNA binding motif of a sequence-specific DNA binding protein relative to the DNA site in the protein-DNA complex. The method involves incorporation of a photoactivatable crosslinking agent at a single site within the DNA binding motif of the sequence-specific DNA binding protein, formation of the derivatized protein-DNA complex, UV-irradiation of the derivatized protein-DNA complex, and determination of the nucleotide(s) at which crosslinking occurs. We have applied the method to catabolite gene activator protein (CAP). We have constructed and analyzed two derivatives of CAP: one having a phenyl azide photoactivatable crosslinking agent at amino acid 2 of the helix-turn-helix motif of CAP, and one having a phenyl azide photoactivatable crosslinking agent at amino acid 10 of the helix-turn-helix motif of CAP. The results indicate that amino acid 2 of the helix-turn-helix motif is close to the top-strand nucleotides of base pairs 3 and 4 of the DNA half site in the CAP-DNA complex, and that amino acid 10 of the helix-turn-helix motif is close to the bottom-strand nucleotide of base pair 10 of the DNA half site in the CAP-DNA complex. The results define unambiguously the orientation of the helix-turn-helix motif relative to the DNA half site in the CAP-DNA complex. Comparison of the results to the crystallographic structure of the CAP-DNA complex [Schultz, S., Shields, S. & Steitz, T. (1991) Science 253, 1001-1007] indicates that the method provides accurate, high-resolution proximity and orientation information.
Ebright, YW, Chen Y, Pendergrast PS, Ebright RH.  1992.  Incorporation of an EDTA-metal complex at a rationally selected site within a protein: application to EDTA-iron DNA affinity cleaving with catabolite gene activator protein (CAP) and Cro.. Biochemistry. 31(44):10664-70. Abstract
We have developed a simple procedure to incorporate an EDTA-metal complex at a rationally selected site within a full-length protein. Our procedure has two steps: In step 1, we use site-directed mutagenesis to introduce a unique solvent-accessible cysteine residue at the site of interest. In step 2, we derivatize the resulting protein with S-(2-pyridylthio)cysteaminyl-EDTA-metal, a novel aromatic disulfide derivative of EDTA-metal. We have used this procedure to incorporate an EDTA-iron complex at amino acid 2 of the helix-turn-helix motif of each of two helix-turn-helix motif sequence-specific DNA binding proteins, catabolite gene activator protein (CAP) and Cro, and we have analyzed EDTA-iron-mediated DNA affinity cleavage by the resulting protein derivatives. The CAP derivative cleaves DNA at base pair 2 of the DNA half-site in the protein-DNA complex, and the Cro derivative cleaves DNA at base pairs -3 to 5 of the DNA half-site in the protein-DNA complex. We infer that amino acid 2 of the helix-turn-helix motif of CAP is close to base pair 2 of the DNA half-site in the CAP-DNA complex in solution and that amino acid 2 of the helix-turn-helix motif of Cro is close to base pairs -3 to 5 of the DNA half-site in the Cro-DNA complex in solution.(ABSTRACT TRUNCATED AT 250 WORDS)