CAP-DNA complex

Many biochemical and biophysical studies of CAP-DNA complexes in solution have demonstrated that CAP induces a sharp bend in DNA upon binding. This was confirmed when the group of Thomas Steitz at Yale University determined the crystal structure of cyclic AMP-DNA complex to 3 A resolution. The CAP molecule comprises two identical polypeptide chains of 209 amino acid residues (Figure 8.24). Each chain is folded into two domains that have separate functions (Figure 8.24b). The larger N-terminal domain binds the allosteric effector molecule, cyclic AMP, and provides all the subunit interactions that form the dimer. The C-terminal domain contains the helix-tum-helix motif that binds DNA. 

Fried, M. G., and Liu, G. (1994). Molecular sequestration stabilizes CAP—DNA complexes during polyacrylamide gel electrophoresis. Nucleic Acids Res. 22, 5054—5059. 

Earlier work indicated that in the specific CAP-DNA complex the DNA was bent 90° away from the protein but was not distorted in the nonspecific complex. (CAP bound to its noncognate DNA site) [3, 36], This DNA bending phenomenon was exploited to construct a new CAP-OP nuclease capable of single site cleavage [37]. The crystal structure of the specific CAP-DNA complex revealed that amino acids 24-26 and 89-91 of CAP were close to the DNA substrate [38], With this in mind, residue 26 was mutated... 

Figure 4. Structure of the CAP-DNA complex shows angles ofDNA bending in the CAP-DNA complex. The DNA helix axis as defined by the program Curves is shown as a black line running down the middle of the DNA helix. (Adapted with permission from reference 17)...

Tab. 5.3 UV melting data for hydrophobic N-capped B5 aPNA DNA complexes...

The interaction between the CAP-cAMP complex and DNA is shown in Fig. 4. Each subunit of the dimeric inducer (yellow or orange) binds one molecule of cAMP (red). Contact with the DNA (blue) is mediated by two recognition helices that interact with the major groove of the DNA. The bending of the DNA strand caused by CAP has functional significance. 

Fig. 1.15. Bending of the DNA in the CAP protein-DNA complex. The CAP protein ( . coli) binds as a dimer to the two-fold symmetric operator sequence. The DNA is bent nearly 90deg in the complex. The turns are centered around two GT sequences (shown in black) of the recognition element.

The CAP binds to DNA with the consensus sequence 5 -A A ATGTG ATCT/5 -AGATCACATTT, which may be located at variable distances from the promoter.133 How does binding of the CAP-cAMP complex increase the rate of initiation of mRNA transcription The anwer evidently lies in direct interaction between CAP and the N-terminal domain of the RNAP a subunit.54d 129 Binding of CAP induces a 90° bend in the DNA, which may facilitate the protein-protein interaction and may lead to looping.130134... 

A transcription-regulating protein that binds to DNA in the promoter loop. See Benoff, B., Yang, H., Lawson, C.L. et al.. Structural basis of transcription activation the CAP-alpha CTD-DNA complex. Science 297, 1562-1566, 2002 Balaeff, A., Madadevan, L., and Schulten, K., Structural basis for cooperative DNA binding by CAP and lac repressor. Structure 12, 123-132, 2004 Akaboshi, E., Dynamic profiles of DNA analysis of CAP-and LexA protein-binding regions with endonucleases, DNA Cell Biol. 24, 161-172, 2005. 

The ara operon is positively controlled by the activity of AraC, which can act both as a repressor and activator of ara operon transcription, depending on the intracellular concentrations of arabinose and glucose. In the absence of arabinose, AraC is a repressor and binds to two separate operator sites in the ara operon, leading to the formation of an inhibitory DNA loop. However, in the presence of arabinose, which binds to AraC, and in the presence of CAP-cAMP complexes, the inhibitory DNA loop is disrupted and CAP-cAMP complexes are able to stimulate transcription of the ara operon. 

Figure 30 Chemical structure of PDFD, schematics of a TGA (thioglycolic acid)-capped CdTe QD and of the PDFD/QD/dye-labeled DNA (IRD700-labeled ds-DNA) complex used to detect DNA hybridization through a cascaded double FRET. Upon optical excitation of PDFD and QDs, energy transfer takes place from the PDFD to the QDs (FRET 1) and from the QDs to the dye-labeled DNA (FRET 2).

Figure 34 Mixed representation of the p53 DNA complex. The p53 protein and the DNA is shown as capped stick model, the protein backbone is represented as ribbon model. Parts of the molecular surface indicate the p53 protein DNA interface region. The surfaces are color coded with respect to the electrostatic potential calculated by a finite difference algorithm solving the Poisson-Boltzmann equation - (blue, negative gray, neutral red. positive). The electropositive parts of the p53 protein fit perfectly in the major and minor groove of the almost electronegative DNA...

Figure 41 VRML scene of the mutated p53 DNA complex. The DNA fragment is shown in wire frame representation, the backbone of the protein in ribbon representation with the mutation hotspots in capped sticks (magenta, mutated amino acid ARG248 > GLN248). The space button in the lower left comer leads to a lower quality representation of this scene...

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