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CALTECH APH 161 - Structural Basis of Transcription Activation

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Structural Basis of TranscriptionActivation: The CAP-␣CTD-DNAComplexBrian Benoff,1Huanwang Yang,1Catherine L. Lawson,1Gary Parkinson,1* Jinsong Liu,1† Erich Blatter,1,2‡Yon W. Ebright,1,2Helen M. Berman,1§ Richard H. Ebright1,2㛳The Escherichia coli catabolite activator protein (CAP) activates transcription atPlac,Pgal, and other promoters through interactions with the RNA polymerase␣ subunit carboxyl-terminal domain (␣CTD). We determined the crystal struc-ture of the CAP-␣CTD-DNA complex at a resolution of 3.1 angstroms. CAPmakes direct protein-protein interactions with ␣CTD, and ␣CTD makes directprotein-DNA interactions with the DNA segment adjacent to the DNA site forCAP. There are no large-scale conformational changes in CAP and ␣CTD, andthe interface between CAP and ␣CTD is small. These findings are consistent withthe proposal that activation involves a simple “recruitment” mechanism.The catabolite activator protein (CAP) [alsoreferred to as the cyclic adenosine monophos-phate (cAMP) receptor protein] activatestranscription by binding to a DNA site locat-ed in or upstream of the core promoter andinteracting with the RNA polymerase(RNAP) ␣ subunit COOH-terminal domain(␣CTD) [reviewed in (1)], an 85–amino acid,independently folded domain that is flexiblytethered to the remainder of RNAP [reviewedin (2)]. Interaction of CAP with ␣CTD facil-itates binding of ␣CTD (and, through it, theremainder of RNAP) to promoter DNA,thereby stimulating transcription initiation(1). At class I CAP-dependent promoters,such as the Placpromoter, CAP-␣CTD inter-action is the sole basis of activation (1). Atclass II CAP-dependent promoters, such asthe Pgalpromoter, interaction of CAP with␣CTD is one of multiple interactions in-volved in activation (1, 3).CAP binds to DNA as a dimer of twoidentical subunits and recognizes a 22– basepair (bp), two-fold symmetric consensusDNA site (1, 4–6). Transcription activationby CAP requires a determinant within CAPtermed “activating region 1” (AR1) (residues156 to 164) (1, 7–11), which is functionallypresented by one of the two subunits of theCAP dimer (1, 12, 13). Transcription activa-tion also requires the COOH-terminal residueof CAP (residue 209) (14), which, in thestructure of the CAP-DNA complex, is locat-ed adjacent to, and is in contact with, AR1(6).Transcription activation by CAP requiresthree distinct determinants within ␣CTD (1,15–19): (i) the “287 determinant” (residues285 to 290, 315, 317, and 318), proposed tomediate protein-protein interaction with AR1of CAP; (ii) the “265 determinant” (residues265, 294, 296, 298, 299, and 302), proposedto mediate protein-DNA interaction with theDNA segment adjacent to the DNA site forCAP; and (iii) the “261 determinant” (resi-dues 257, 258, 259, and 261), proposed tomediate protein-protein interaction with ␴70at a subset of class I CAP-dependent promot-ers, including Plac.Transcription activation by CAP also re-quires the structural integrity of the DNAsegment adjacent to the DNA site for CAP (1,20). In the ternary complex of CAP, RNAP,and promoter, ␣CTD interacts with the DNAsegment adjacent to the DNA site for CAP,contacting the DNA minor groove centered18 or 19 bp from the center of the DNA sitefor CAP (1, 21, 22). At most CAP-dependentpromoters, including Plac, ␣CTD interactsnonspecifically with the DNA segment adja-cent to the DNA site for CAP, contactingarbitrary, nonspecific DNA sequences (1, 23,24). However, replacement of these nonspe-cific DNA sequences by high-affinity, specif-ic DNA sites for ␣CTD (e.g., 5⬘-AAAAAA-3⬘)(25) facilitates formation of the ternarycomplex of CAP, RNAP, and promoter (1,24, 26 –28).In previous work, the CAP-DNA complexwas crystallized using a 30-bp two-fold sym-metric DNA fragment containing the 22-bptwo-fold symmetric consensus DNA site forCAP and 4 bp of flanking DNA on each side(Fig. 1A, top) [(6); see also (4, 5)]. In ourcurrent work, we crystallized the CAP-␣CTD-DNA complex using an analogous 44-bp two-fold symmetric DNA fragment con-taining the 22-bp two-fold symmetric consen-sus DNA site for CAP and 11 bp of flankingDNA—with an optimally positioned, high-affinity, specific DNA site for ␣CTD (i.e.,5⬘-AAAAAA-3⬘)(25, 27, 28)— on each side(Fig. 1B, top) (29). It was anticipated that thisDNA fragment would yield a two-fold sym-metric complex consisting of a central CAPdimer flanked on each side by ␣CTD (Fig.1B).The structure was solved by molecularreplacement using the crystal structure of theCAP-DNA complex as the initial model anditerative cycles of Fourier refinement andmodel building to place the rest of the struc-ture. The final model R and Rfreevalues are,respectively, 21.1 and 24.4% against 3.1 Ådiffraction data (Fig. 1C) (30).As anticipated, the two-fold symmetriccrystallization DNA fragment yielded a two-fold symmetric structure (Fig. 1B). Each halfof the two-fold symmetric structure containsone subunit of CAP, one-half of the crystal-lization DNA fragment, one molecule of␣CTD that interacts with CAP and DNA(␣CTDCAP,DNA), and one molecule of ␣CTDthat interacts exclusively with DNA, interact-ing with an A/T-rich DNA-minor-groovesegment that, fortuitously, is accessible in thecrystal lattice (␣CTDDNA). We suggest thatthe structure defines two distinct sets of bio-logically relevant interactions: (i) CAP-␣CTD-DNA interactions at a class I or classII CAP-dependent promoter (Fig. 2; CAP-␣CTDCAP,DNA-DNA) and (ii) ␣CTD-DNAinteractions with an A/T-rich DNA minorgroove at an UP element subsite– dependentpromoter [Fig. 3; ␣CTDDNA-DNA (2, 25)].The structures of the CAP-DNA complex(Fig. 1A) (6) and of CAP-DNA within thepresent complex (Fig. 1B) are superimpos-able [root mean square deviation of backboneatoms (RMSDbackbone) ⫽ 1.0 Å]. We con-clude that the conformations of CAP and theDNA segment in contact with CAP do notchange substantially upon interaction with␣CTD. Similarly, the structures of free␣CTD (31), ␣CTDCAP,DNA, and ␣CTDDNAare superimposable (RMSDbackbone⫽ 0.7 to2.1 Å). We conclude that the conformation of␣CTD does not change substantially uponinteraction with CAP and/or with DNA. Thestructure of the DNA segment between CAPand ␣CTDCAP,DNAis moderately distorted(roll of 15° and twist deficit of 10° centered13 bp from the center of the DNA site forCAP, resulting in compression by ⬃2.5Åofthe DNA major groove centered 13 bp fromthe center


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CALTECH APH 161 - Structural Basis of Transcription Activation

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