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Structural Basis of Transcription Activation: The CAP-αCTD-DNA Complex

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Science  30 Aug 2002:
Vol. 297, Issue 5586, pp. 1562-1566
DOI: 10.1126/science.1076376

Abstract

The Escherichia coli catabolite activator protein (CAP) activates transcription at Plac, Pgal, and other promoters through interactions with the RNA polymerase α subunit carboxyl-terminal domain (αCTD). We determined the crystal structure of the CAP-αCTD-DNA complex at a resolution of 3.1 angstroms. CAP makes direct protein-protein interactions with αCTD, and αCTD makes direct protein-DNA interactions with the DNA segment adjacent to the DNA site for CAP. There are no large-scale conformational changes in CAP and αCTD, and the interface between CAP and αCTD is small. These findings are consistent with the proposal that activation involves a simple “recruitment” mechanism.

The catabolite activator protein (CAP) [also referred to as the cyclic adenosine monophosphate (cAMP) receptor protein] activates transcription by binding to a DNA site located in or upstream of the core promoter and interacting with the RNA polymerase (RNAP) α subunit COOH-terminal domain (αCTD) [reviewed in (1)], an 85–amino acid, independently folded domain that is flexibly tethered to the remainder of RNAP [reviewed in (2)]. Interaction of CAP with αCTD facilitates binding of αCTD (and, through it, the remainder of RNAP) to promoter DNA, thereby stimulating transcription initiation (1). At class I CAP-dependent promoters, such as the Placpromoter, CAP-αCTD interaction is the sole basis of activation (1). At class II CAP-dependent promoters, such as the Pgal promoter, interaction of CAP with αCTD is one of multiple interactions involved in activation (1, 3).

CAP binds to DNA as a dimer of two identical subunits and recognizes a 22–base pair (bp), two-fold symmetric consensus DNA site (1,4–6). Transcription activation by CAP requires a determinant within CAP termed “activating region 1” (AR1) (residues 156 to 164) (1, 7–11), which is functionally presented by one of the two subunits of the CAP dimer (1, 12, 13). Transcription activation also requires the COOH-terminal residue of CAP (residue 209) (14), which, in the structure of the CAP-DNA complex, is located adjacent to, and is in contact with, AR1 (6).

Transcription activation by CAP requires three distinct determinants within αCTD (1, 15–19): (i) the “287 determinant” (residues 285 to 290, 315, 317, and 318), proposed to mediate protein-protein interaction with AR1 of CAP; (ii) the “265 determinant” (residues 265, 294, 296, 298, 299, and 302), proposed to mediate protein-DNA interaction with the DNA segment adjacent to the DNA site for CAP; and (iii) the “261 determinant” (residues 257, 258, 259, and 261), proposed to mediate protein-protein interaction with σ70 at a subset of class I CAP-dependent promoters, including Plac.

Transcription activation by CAP also requires the structural integrity of the DNA segment adjacent to the DNA site for CAP (1, 20). In the ternary complex of CAP, RNAP, and promoter, αCTD interacts with the DNA segment adjacent to the DNA site for CAP, contacting the DNA minor groove centered 18 or 19 bp from the center of the DNA site for CAP (1, 21, 22). At most CAP-dependent promoters, including Plac, αCTD interacts nonspecifically with the DNA segment adjacent to the DNA site for CAP, contacting arbitrary, nonspecific DNA sequences (1, 23, 24). However, replacement of these nonspecific DNA sequences by high-affinity, specific DNA sites for αCTD (e.g., 5′-AAAAAA-3′) (25) facilitates formation of the ternary complex of CAP, RNAP, and promoter (1, 24, 26–28).

In previous work, the CAP-DNA complex was crystallized using a 30-bp two-fold symmetric DNA fragment containing the 22-bp two-fold symmetric consensus DNA site for CAP and 4 bp of flanking DNA on each side (Fig. 1A, top) [(6); see also (4, 5)]. In our current work, we crystallized the CAP-αCTD-DNA complex using an analogous 44-bp two-fold symmetric DNA fragment containing the 22-bp two-fold symmetric consensus DNA site for CAP and 11 bp of flanking DNA—with an optimally positioned, high-affinity, specific DNA site for αCTD (i.e., 5′-AAAAAA-3′) (25, 27, 28)—on each side (Fig. 1B, top) (29). It was anticipated that this DNA fragment would yield a two-fold symmetric complex consisting of a central CAP dimer flanked on each side by αCTD (Fig. 1B).

Figure 1

Structure determination. (A) Top, 30-bp DNA fragment used to crystallize the CAP-DNA complex in (6) (box, DNA site for CAP; solid circle, two-fold symmetry axis of DNA site for CAP; solid bars, single-phosphate gaps). Bottom, structure of the CAP-DNA complex in (6). CAP is in cyan; DNA and cAMP are in red. (B) Top, 44-bp DNA fragment used to crystallize the CAP-αCTD-DNA complex in this work (box, DNA site for CAP; solid circle, two-fold symmetry axis of DNA site for CAP; solid bars, single-phosphate gaps). Bottom, structure of the CAP-αCTD-DNA complex of this work. CAP is in cyan, αCTDCAP,DNA is in light green, αCTDDNA is in dark green, and DNA and cAMP are in red. (C) Crystallographic data collection and refinement statistics. I, intensity.

The structure was solved by molecular replacement using the crystal structure of the CAP-DNA complex as the initial model and iterative cycles of Fourier refinement and model building to place the rest of the structure. The final model R andR free values are, respectively, 21.1 and 24.4% against 3.1 Å diffraction data (Fig. 1C) (30).

As anticipated, the two-fold symmetric crystallization DNA fragment yielded a two-fold symmetric structure (Fig. 1B). Each half of the two-fold symmetric structure contains one subunit of CAP, one-half of the crystallization DNA fragment, one molecule of αCTD that interacts with CAP and DNA (αCTDCAP,DNA), and one molecule of αCTD that interacts exclusively with DNA, interacting with an A/T-rich DNA-minor-groove segment that, fortuitously, is accessible in the crystal lattice (αCTDDNA). We suggest that the structure defines two distinct sets of biologically relevant interactions: (i) CAP-αCTD-DNA interactions at a class I or class II CAP-dependent promoter (Fig. 2; CAP-αCTDCAP,DNA-DNA) and (ii) αCTD-DNA interactions with an A/T-rich DNA minor groove at an UP element subsite–dependent promoter [Fig. 3; αCTDDNA-DNA (2, 25)].

Figure 2

CAP-αCTDCAP,DNA-DNA(interactions representative of those at a class I or class II CAP-dependent promoter). (A) Stereo view of interactions among CAP, αCTDCAP,DNA, and DNA (two orthogonal views). AR1 of CAP is in blue; the 287 determinant (CAP contact), 265 determinant (DNA contact), and the 261 determinant (proposed σ70 contact) of αCTDCAP,DNA are in yellow, red, and gray-white, respectively. (B) Interactions between AR1 of CAP and residues 285 to 288 of the 287 determinant of αCTDCAP,DNA. Hydrogen bonds are in magenta. (C) Interactions between the COOH-terminal residue of CAP (Arg209) and residues 315 and 317 of the 287 determinant of αCTDCAP,DNA. Hydrogen bonds are in magenta. C-TER, COOH-terminus. (D) Interactions between αCTDCAP,DNA and DNA (view along DNA minor-groove axis). Water-mediated hydrogen bonds involving the Arg265side-chain guanidinium, DNA bases, and an experimentally defined water molecule (sphere near center) are in cyan. The network of hydrogen bonds buttressing the Arg265 side-chain guanidinium relative to the phosphate backbones of the two DNA strands is in yellow. Other hydrogen bonds are in magenta. (E) Summary of interactions between αCTDCAP,DNA and DNA. Colors are as in (D). G, Gly; K, Lys; N, Asn; R, Arg; S, Ser; and V, Val.

Figure 3

αCTDDNA-DNA (interactions representative of those at an UP element subsite–dependent promoter). (A) Stereo view comparing interactions between αCTDDNA and DNA (dark green and gray) and interactions between αCTDCAP-DNA and DNA (light green and gray) (RMSD = 0.74 Å for 72 Cα and 10 P atoms). (B) Interactions between αCTDDNAand DNA. View and colors are as in Fig. 2D. No water molecules were observed in the αCTDDNA-DNA interface in this structure at 3.1 Å. However, the positions of the Arg265side-chain guanidinium and DNA bases are compatible with the establishment of water-mediated hydrogen bonds identical to those at the αCTDCAP,DNA-DNA interface (Fig. 2D) (39). (C) Summary of interactions between αCTDDNA and DNA. Colors are as in Fig. 2, D and E.

The structures of the CAP-DNA complex (Fig. 1A) (6) and of CAP-DNA within the present complex (Fig. 1B) are superimposable [root mean square deviation of backbone atoms (RMSDbackbone) = 1.0 Å]. We conclude that the conformations of CAP and the DNA segment in contact with CAP do not change substantially upon interaction with αCTD. Similarly, the structures of free αCTD (31), αCTDCAP,DNA, and αCTDDNA are superimposable (RMSDbackbone = 0.7 to 2.1 Å). We conclude that the conformation of αCTD does not change substantially upon interaction with CAP and/or with DNA. The structure of the DNA segment between CAP and αCTDCAP,DNA is moderately distorted (roll of 15° and twist deficit of 10° centered 13 bp from the center of the DNA site for CAP, resulting in compression by ∼2.5 Å of the DNA major groove centered 13 bp from the center of the DNA site for CAP, and reduction by ∼7.5 Å of the distance between CAP and αCTDCAP,DNA) (32, 33). The structures of the DNA segments in contact with αCTDCAP,DNA and αCTDDNA are nearly canonical B-form DNA, with only very slight distortion (compression by ∼1.5 Å of the DNA minor groove).

The interaction between CAP and αCTDCAP,DNAinvolves AR1 and the COOH-terminal residue of CAP and the 287 determinant of αCTDCAP,DNA (Fig. 2, A through C). The interaction involves a strikingly small interface (six residues each of CAP and αCTD; 630 Å2 of buried surface area). Residues 157 to 160 and 164 of AR1 contact residues 285 to 288 of the 287 determinant, with the Thr158 side-chain hydroxyl making two hydrogen bonds (to residues 285 and 286), the Thr158backbone carbonyl making two hydrogen bonds (to residues 285 and 287), the Thr158 side-chain methyl making van der Waals interactions (to residue 286), and residues 157, 159, 160, and 164 making additional van der Waals interactions (Fig. 2B). The COOH-terminal residue of CAP (Arg209) contacts residues 315 and 317 of the 287 determinant of αCTD, with the COOH-terminal residue side chain making a hydrogen bond to residue 315 and the COOH-terminal backbone carboxylate making a salt bridge with residue 317 (Fig. 2C). The COOH-terminal residue side chain also makes two buttressing hydrogen bonds to AR1 (to backbone carbonyl oxygens of residues 159 and 160). The observed contacts provide a structural rationalization for genetic results indicating that Thr158is the critical residue in AR1 (the only residue in AR1 for which side-chain atoms beyond Cβ are required for activation) (10) and indicating involvement of residues 157 to 160, 164, and the COOH-terminus of CAP, and residues 285 to 288, 315, and 317 of αCTD (1, 7–11, 14, 18, 19). The structure also provides a rationalization for genetic results indicating involvement of residues 162 and 163 of CAP and residues 289 and 290 of αCTD (1, 8–11,19); in the structure, these residues underlie (and make hydrogen bonds and van der Waals interactions with) residues of AR1 and the 287 determinant of αCTD and, as such, are likely to be critical determinants of the conformations of AR1 and the 287 determinant. The observed small size of the interface between CAP and αCTDCAP,DNA is in agreement with genetic results ruling out involvement of other residues (10, 18,19) and with biochemical results indicating a modest magnitude of transcriptional activation (a factor of about 10 at Plac) (34) and a modest magnitude of CAP-RNAP interaction free energy (about –1 to –2 kcal/mol at Plac) (35–37).

The interaction between αCTDCAP,DNA and DNA involves the 265 determinant of αCTDCAP,DNA and the DNA backbone and minor groove of the 6-bp DNA segment centered 19 bp from the center of the DNA site for CAP (5′-AAAAAG-3′) (Fig. 2, A, D, and E). Residues 264, 265, 268, 294, 296, 298, and 299 of the 265 determinant make direct contacts with the DNA backbone (Fig. 2, D and E). No residue makes direct contacts with DNA base-pair edges (Fig. 2, D and E). However, the Arg265 side chain penetrates into the DNA minor groove and makes at least two, and possibly four, water-mediated hydrogen bonds with DNA base-pair edges (cyan in Fig. 2, D and E). The Arg265 side-chain guanidinium makes two water-mediated hydrogen bonds (to the thymine O2 atom of the A:T base pair 18.5 bp from the center of the DNA site for CAP, and to the adenine N3 atom of the A:T base pair 19.5 bp from the center of the DNA site for CAP) through interaction with a water molecule positioned as in the “minor-groove spine of water” characteristic of A/T-rich DNA (38–41) (cyan in Fig. 2, D and E). In addition, the Arg265 side-chain guanidinium is positioned so as potentially to make two additional water-mediated hydrogen bonds (to the thymine O2 atom of the A:T base pair 19.5 bp from the center of the DNA site for CAP, and to the adenine N3 atom of the A:T base pair 20.5 bp from the center of the DNA site for CAP) through interaction with a putative second water molecule positioned as in the minor-groove spine of water (a water molecule not observed in this structure at 3.1 Å) (39). The Arg265 side-chain guanidinium is held in a precise orientation relative to the DNA minor groove and bound water through buttressing van der Waals interactions with DNA-backbone sugars of the top and bottom DNA strands (Fig. 2D) and by a cross-helix network of buttressing hydrogen bonds, involving two hydrogen bonds between the Arg265 side-chain guanidinium and Asn294, one hydrogen bond between Asn294and a DNA-backbone phosphate of the top DNA strand, and one hydrogen bond between Asn294 and a DNA-backbone phosphate of the bottom DNA strand (yellow in Fig. 2, D and E). The observed contacts provide a structural rationalization for genetic and nuclear magnetic resonance (NMR)–spectroscopic results indicating involvement of residues 264, 265, 268, 294, 296, 298, and 299 (with Arg265as the most critical residue) in CAP-αCTD-DNA and αCTD-DNA interactions (1, 15–19, 25, 42, 43), for biochemical and NMR-spectroscopic results indicating involvement of the DNA minor groove in CAP-αCTD-DNA and αCTD-DNA interactions (22, 43,44), and for interference-footprinting results indicating involvement of the adenine N3 atom in αCTD-DNA interactions (44). The observed contacts also provide two structural rationalizations for the specificity of αCTD for A/T-rich DNA: (i) Arg265 makes water-mediated hydrogen bonds through interaction with the minor-groove spine of water, a structural feature present in A/T-rich DNA (40, 41), and (ii) Arg265 is buttressed by a network of interactions spanning a compressed, narrowed minor groove, a structural feature present in A/T-rich DNA (45, 46).

The 261 determinant of αCTDCAP,DNA is located on the face of αCTDCAP,DNA opposite from CAP, ∼23 bp from the center of the DNA site for CAP, and is prominently exposed (gray-white in Fig. 2A). The position and prominent exposure of the 261 determinant are consistent with the proposal that the 261 determinant participates in αCTD-σ70 interactions at the subset of class I CAP-dependent promoters, including Plac, where CAP binds in the –60 region, with ∼23 bp between the center of the DNA site for CAP and the upstream edge of the DNA segment contacted by σ70 (1, 19, 47).

As noted above, the crystal structure contains a second molecule of αCTD that interacts exclusively with DNA, interacting with an UP element subsite–like DNA segment (5′-GAAAAA-3′) [compare with (25)] that, fortuitously, is accessible in the crystal lattice: αCTDDNA (dark green in Fig. 1B; Fig. 3). αCTDDNA-DNA and αCTDCAP,DNA-DNA interactions are nearly identical, both in overall organization and in detail (Fig. 3). We infer that similar αCTD-DNA interactions are made in UP element subsite–dependent transcription and in CAP-dependent transcription, consistent with similar requirements for the 265 determinant (1, 15–19, 25, 42, 43) and similar preferences for A/T-rich DNA (24–28).

The results in this report provide a high-resolution structural description of interactions between a transcriptional activator and its target within the general transcription machinery. Two striking findings are that transcriptional activation can occur without conformational change in activator and target and that transcriptional activation can involve a small interface between activator and target. These findings support the proposal that transcriptional activation can involve a simple “recruitment” mechanism—that is, simple “adhesive” interactions between activator and target that facilitate and/or stabilize interaction of the general transcription machinery with promoter DNA (1, 2, 48–50). Activation by recruitment does not require conformational signaling within or through the target; does not require extensive, high-information-content interactions between activator and target; and entails modest net interaction energies between activator and target (interaction energies comparable to the magnitude of activation) (48–50). We suggest that the results provide a structural paradigm for understanding other examples of transcriptional activation, both in bacteria (where most activators are thought to function through recruitment and to contact the same target, αCTD) (1, 2,48–50) and in eukaryotes (where most activators are thought to function through recruitment) (49, 50).

Supporting Online Material

www.sciencemag.org/cgi/content/full/297/5586/1562/DC1

Figs. S1 and S2

  • * Present address: CRC Biomolecular Structure Unit, Institute of Cancer Research, London SW3 6JB, UK.

  • Present address: Tularik Inc., South San Francisco, CA 94080, USA.

  • Present address: Human Genome Sciences Inc., Rockville, MD 20874, USA.

  • § To whom correspondence on crystallographic issues should be addressed. E-mail: berman{at}rutchem.rutgers.edu

  • || To whom correspondence on all other issues should be addressed. E-mail: ebright{at}mbcl.rutgers.edu

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