Charge neutralization and DNA bending by the Escherichia coli catabolite activator protein

We are interested in the role of asymmetric phosphate neutralization in DNA bending induced by proteins. We describe an experimental estimate of the actual electrostatic contribution of asymmetric phosphate neutralization to the bending of DNA by the Escherichia coli catabolite activator protein (CAP), a prototypical DNA-bending protein. Following assignment of putative electrostatic interactions between CAP and DNA phosphates based on X-ray crystal structures, appropriate phosphates in the CAP half-site DNA were chemically neutralized by methylphosphonate substitution. DNA shape was then evaluated using a semi-synthetic DNA electrophoretic phasing assay. Our results confirm that the unmodified CAP DNA half-site sequence is intrinsically curved by 26° in the direction enhanced in the complex with protein. In the absence of protein, neutralization of five appropriate phosphates increases DNA curvature to 32° (~23% increase), in the predicted direction. Shifting the placement of the neutralized phosphates changes the DNA shape, suggesting that sequence-directed DNA curvature can be modified by the asymmetry of phosphate neutralization. We suggest that asymmetric phosphate neutralization contributes favorably to DNA bending by CAP, but cannot account for the full DNA deformation.     

A model for the non-specific binding of catabolite gene activator protein to DNA

The binding of E. coli catabolite gene activator protein (CAP) to non-specific sequences of DNA has been modelled as an electrostatic interaction between four basic side chains of the CAP dimer and the charged phosphates of DNA. Calculation of the electrostatic contribution to the binding free energy at various separations of the two molecules shows that complex formation is favored when CAP and DNA are separated by as much as 12 A. Thus, the long range electrostatic interactions may provide the initial energy for complex formation and also the correct relative orientation of CAP and DNA. The non-specific complex does not involve the penetration of amino acid side chains into the major grooves of DNA and permits 'sliding' of the protein along DNA, which would enhance the rate of association of CAP with the specific site as has been proposed previously for lac repressor. We propose that, as it 'slides', CAP is moving in and out of the major grooves in order to sample the DNA sequence. Recognition of the specific DNA site is achieved by a complementarity in structure and hydrogen bonding between amino acids and the edges of base pairs exposed in the major grooves of DNA.     

DNA sequence determinants for binding of the Escherichia coli catabolite gene activator protein.

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.     

The energetic contribution of induced electrostatic asymmetry to DNA bending by a site-specific protein

DNA bending can be promoted by reducing the net negative electrostatic potential around phosphates on one face of the DNA, such that electrostatic repulsion among phosphates on the opposite face drives bending toward the less negative surface. To provide the first assessment of energetic contribution to DNA bending when electrostatic asymmetry is induced by a site-specific DNA binding protein, we manipulated the electrostatics in the EcoRV endonuclease-DNA complex by mutation of cationic side chains that contact DNA phosphates and/or by replacement of a selected phosphate in each strand with uncharged methylphosphonate. Reducing the net negative charge at two symmetrically located phosphates on the concave DNA face contributes − 2.3 kcal mol^− 1 to − 0.9 kcal mol^− 1 (depending on position) to complex formation. In contrast, reducing negative charge on the opposing convex face produces a penalty of + 1.3 kcal mol^− 1. Förster resonance energy transfer experiments show that the extent of axial DNA bending (about 50°) is little affected in modified complexes, implying that modification affects the energetic cost but not the extent of DNA bending. Kinetic studies show that the favorable effects of induced electrostatic asymmetry on equilibrium binding derive primarily from a reduced rate of complex dissociation, suggesting stabilization of the specific complex between protein and markedly bent DNA. A smaller increase in the association rate may suggest that the DNA in the initial encounter complex is mildly bent. The data imply that protein-induced electrostatic asymmetry makes a significant contribution to DNA bending but is not itself sufficient to drive full bending in the specific EcoRV-DNA complex.     

Crystal structure of a cyclic AMP-independent mutant of catabolite gene activator protein

Escherichia coli NCR91 synthesizes a mutant form of catabolite gene activator protein (CAP) in which alanine 144 is replaced by threonine. This mutant, which also lacks adenylate cyclase activity, has a CAP phenotype; in the absence of cAMP it is able to express genes that normally require cAMP. CAP91 has been purified and crystallized with cAMP under the same conditions as used to crystallize the wild type CAP X cAMP complex. X-ray diffraction data were measured to 2.4-A resolution and the CAP91 structure was determined using initial model phases from the wild type structure. A difference Fourier map calculated between CAP91 and wild type showed the 2 alanine to threonine sequence changes in the dimer and also a change in orientation of cysteine 178 in one of the subunits. The CAP91 coordinates were refined by restrained least squares to an R factor of 0.186. Differences in the atomic positions of the wild type and mutant protein structures were analyzed by a vector averaging technique. There were small changes that included concerted motions in the small domains, in the hinge between the two domains and in an adjacent loop between beta-strands 4 and 5. The mutation at residue 144 apparently causes changes in the position of some protein atoms that are distal to the mutation site.     

DNA bending of pSC101 ori induced by Rep,a replication initiator protein and IHF

Purified Rep (or RepA) protein, a replication initiator of plasmid pSC101, is present almost solely in the dimer form, and its binding activity for the directly repeated sequences (iterons) in the replication origin (ori) is very low. When Rep protein was treated with guanidine hydrochloride followed by renaturation, it was shown to bind to the iterons with very high efficiency. A gel shift experiment suggested that guanidine-treated Rep bound to iterons as a monomer form. The Rep monomer bound noncooperatively to the three iterons and induced bending of the DNA helix axis in the same direction (about 100 degrees ). The configuration of the IHF box that is a binding site of another DNA bending protein IHF, the three iterons and an AT-rich region between these sequences was important for efficient bending of the ori region. Furthermore, a mutant Rep protein (Rep(IHF)) which can support the plasmid replication in IHF-deficient host cells was purified, and it was found that affinity of the Rep(IHF) monomer for iterons was similar to that of wild-type Rep and bent DNA only 14 degrees more strongly than did the wild-type Rep. Rep(IHF)-dependent plasmid replication, however, required both enhancer regions, par and IR-1, in addition to "core ori" as a minimal essential ori, whereas only one of these two enhancers was necessary for wild-type Rep-dependent replication. How Rep(IHF) can support plasmid replication in the absence of IHF is discussed.     

Crystallization of Escherichia coli catabolite gene activator protein with its DNA binding site. The use of modular DNA

To obtain crystals of the Escherichia coli catabolite gene activator protein (CAP) complexed with its DNA-binding site, we have searched for crystallization conditions with 26 different DNA segments greater than or equal to 28 base-pairs in length that explore a variety of nucleotide sequences, lengths, and extended 5' or 3' termini. In addition to utilizing uninterrupted asymmetric lac site sequences, we devised a novel approach of synthesizing half-sites that allowed us to efficiently generate symmetric DNA segments with a wide variety of extended termini and lengths in the large size range (greater than or equal to 28 bp) required by this protein. We report three crystal forms that are suitable for X-ray analysis, one of which (crystal form III) gives measurable diffraction amplitudes to 3 A resolution. Additives such as calcium, n-octyl-beta-D-glucopyranoside and spermine produce modest improvements in the quality of diffraction from crystal form III. Adequate stabilization of crystal form III is unexpectedly complex, requiring a greater than tenfold reduction in the salt concentration followed by addition of 2-methyl-2,4-pentanediol and then an increase in the concentration of polyethylene glycol.     

Intracellular location of catabolite activator protein of Escherichia coli.

The catabolite activator protein was assayed in extracts from the minicell-producing Escherichia coli strain P678-54. The level of catabolite activator protein was found to be the same in both parent cells and purified minicells, regardless of whether the bacteria were grown on glucose (which leads to low intracellular cyclic adenosine monophosphate levels) or on glycerol-yeast extract or LB broth (which lead to high cyclic adenosine monophosphate concentrations in the cell). Thus, at any given time most catabolite activator protein molecules are found in the cytoplasm. The implications of this for the mechanism of catabolite activator protein action at catabolite-sensitive operons are discussed.     

DNA bending induced by DNA (cytosine-5) methyltransferases

DNA bending induced by six DNA (cytosine-5) methyltransferases was studied using circular permutation gel mobility shift assay. The following bend angles were obtained: M.BspRI (GG^m5CC), 46–50°; M.HaeIII (GG^m5CC), 40–43°; M.SinI (GGW^m5CC), 34–37°; M.Sau96I (GGN^m5CC), 52–57°; M.HpaII (C^m5CGG), 30°; and M.HhaI (G^m5CGC), 13°. M.HaeIII was also tested with fragments carrying a methylated binding site, and it was found to induce a 32° bend. A phase-sensitive gel mobility shift assay, using a set of DNA fragments with a sequence-directed bend and a single methyltransferase binding site, indicated that M.HaeIII and M.BspRI bend DNA toward the minor groove. The DNA curvature induced by M.HaeIII contrasts with the lack of DNA bend observed for a covalent M.HaeIII–DNA complex in an earlier X-ray study. Our results and data from other laboratories show a correlation between the bending properties and the recognition specificities of (cytosine-5) methyltransferases: enzymes recognizing a cytosine 3′ to the target cytosine tend to induce greater bends than enzymes with guanine in this position. We suggest that the observed differences indicate different mechanisms employed by (cytosine-5) methyltransferases to stabilize the helix after the target base has flipped out.     

Linker DNA bending induced by the core histones of chromatin

We have previously reported that ionic conditions that stabilize the folding of long chromatin into 30-nm filaments cause linker DNA to bend, bringing the two nucleosomes of a dinucleosome into contact [Yao, J., Lowary, P. T., & Widom, J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7603-7607]. Dinucleosomes are studied because they allow the unambiguous detection of linker DNA bending through measurement of their nucleosome-nucleosome distance. Because of the large resistance of DNA to bending, the observed compaction must be facilitated by the histones. We have now tested the role of histone H1 (and its variant, H5) in this process. We find that dinucleosomes from which the H1 and H5 have been removed are able to compact to the same extent as native dinucleosomes; the transition is shifted to higher salt concentrations. We conclude that histone H1 is not essential for compacting the chromatin filament. However, H1 contributes to the free energy of compaction, and so it may select a single, ordered, compact state (the 30-nm filament, in long chromatin) from a family of compact states which are possible in its absence.     

Asymmetric DNA bending induced by the yeast multifunctional factor TUF

TUF is a yeast regulatory factor that binds to conserved DNA sequence elements involved in gene activation or silencing as well as in telomere function. Using gel electrophoresis analyses, we show here that TUF induces DNA bending at a site located upstream of the recognition sequence (rpg box). Several point mutations in the rpg box reduced TUF binding strength without affecting the extent of bending. Selective proteolysis of TUF.DNA complexes further suggested the existence of two separate protein domains involved in DNA bending and specific DNA recognition. DNA bending may be an important feature of multifunctional factors that could help them to recruit other proteins for the formation of multiprotein complexes.