A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system.

The use of gel electrophoresis for quantitative studies of DNA-protein interactions is described. This rapid and simple technique involves separation of free DNA from DNA-protein complexes based on differences in their electrophoretic mobilities in polyacrylamide gels. Under favorable conditions both unbound DNA and DNA associated with protein can be quantified. This gel method is applied to the study of the E. coli lactose operon regulatory system. At ionic strengths in the physiological range, the catabolite activator protein (CAP) is shown to form a long-lived complex with the wild type lac promotor, but not with a CAP-insensitive mutant. Formation of a stable "open" or "melted-in" complex of RNA polymerase with the wild type promoter requires the participation of CAP and cyclic AMP. Further, it is demonstrated that even when pre-formed in the presence of CAP-cAMP, the polymerase-promoter open complex becomes unstable if CAP is then selectively removed.     

A new DNA binding mode for CAP

In the absence of cyclic AMP, the Escherichia coli cyclic AMP receptor protein (CAP) binds without detectable sequence specificity to restriction fragments containing lac and crp promoter sequences. Under standard conditions (10 mM Tris, 1 mM EDTA, pH 8.0), our estimates of the equilibrium constant and cooperativity parameter for complex formation are 114,000 +/- 1400 M-1 and 1.3 +/- 0.8, respectively. Thus, this interaction lacks the substantial cooperativity previously reported for CAP binding to genomic DNAs. Using the electrophoresis mobility shift assay, we find that complexes of increasing CAP content differ by a highly uniform mobility decrement. This result is most consistent with a binding mode in which little or no DNA bending occurs. The ability of CAP to distinguish between restriction fragments and genomic DNA, shown by the difference in binding cooperativity, suggests the existence of previously unsuspected DNA sequences or structures that modulate its binding cooperativity.     

The interaction of RNA polymerase and lac repressor with the lac control region.

We have examined the interactions of lac repressor and RNA polymerase with the DNA of the lac control region, using a method for direct visualization of the regions of DNA protected by proteins from DNAase attack. The repressor protects the operator essentially as reported by Gilbert and Maxam (1) with some small modifications. However, the evidence reported here concerning the binding of RNA polymerase to the DNA of the promoter mutant UV5 indicates that : 1) the RNA polymerase molecule binds asymmetrically to the promoter DNA, 2) RNA polymerase protects DNA sequences to within a few bases of the CAP binding site, suggesting direct interaction between polymerase and the CAP protein at this site, 3) RNA polymerase still binds to the promoter when repressor is bound to the operator, but fails to form the same extensive complex.     

Equilibrium studies of the cyclic AMP receptor protein-DNA interaction

The binding of the Escherichia coli cyclic AMP receptor protein (CAP) to restriction fragments containing the lac promoter-operator region has been investigated as a function of cAMP concentration, using a sensitive gel electrophoresis assay. Under standard conditions (13 mM ionic strength), the equilibrium constant for CAP binding to its primary site on a 203 base-pair lac promoter fragment is 6.3 X 10(8) M-1 at 0.2 microM-cAMP, and increases to 8.4 X 10(10) M-1 at 5.0 microM-cAMP. The latter is about 10(5) times larger than the equilibrium constant for binding to an isolated, non-specific site. The L8 mutation, which renders the lac promoter unresponsive to CAP in vivo, lowers this binding affinity by five- to tenfold. Analysis of the cAMP dependency of binding over the concentration range of 0.2 microM to 10 microM reveals that uptake of a single equivalent of cAMP is required for site-specific binding. Similarly, the transfer of CAP from a non-specific DNA site to a specific site requires the net uptake of a single molecule of cAMP. In contrast, co-operative non-specific binding to DNA was found to be independent of cAMP concentration with an equilibrium binding constant of 6 X 10(6) M-1. We conclude that the cAMP affinity of the two CAP subunits in the specific promoter complex is not equal, and that the complex structure therefore deviates significantly from twofold symmetry. A model for the regulation of the lac promoter by the intracellular cAMP concentration is proposed on the basis of the equilibrium binding results.     

Biogenesis of p53 involves cotranslational dimerization of monomers and posttranslational dimerization of dimers. Implications on the dominant negative effect.

Precisely how mutant p53 exerts a dominant negative effect over wild type p53 has been an enigma. To understand how wild type and mutant p53 form hetero-oligomers, we studied p53 biogenesis in vitro. We show here that p53 dimers are formed cotranslationally (on the polysome), whereas tetramers are formed posttranslationally (by the dimerization of dimers in solution). Coexpression of wild type and mutant p53 therefore results in 50% of the p53 generated being heterotetramers comprised of a single species: wild type dimer/mutant dimer. Using hot spot mutants of p53 and a variety of natural target sites, we show that all wild type/mutant heterotetramers manifest impaired DNA binding activity. This impairment is not due to the mutant dimeric subunit inhibiting association of the complex with DNA but rather due to the lack of significant contribution (positive cooperativity) from the mutant partner. For all heterotetramers, bias in binding is particularly pronounced against those sequences in genes responsible for apoptosis rather than cell growth arrest. These results explain the molecular basis of p53 dominant negative effect and suggest a functional role in the regulation of p53 tetramerization.     

Kinetics and mechanism in the reaction of gene regulatory proteins with DNA

We have measured the kinetic properties of the Escherichia coli cAMP receptor protein (CAP) and lac repressor interacting with lac promoter restriction fragments. Under our reaction conditions (10 mM-Tris X HCl (pH 8.0 at 21 degrees C), 1 mM-EDTA, 10 microM-cAMP, 50 micrograms bovine serum albumin/ml, 5% glycerol), the association of CAP is at least a two-step process, with an initial, unstable complex formed with rate constant kappa a = 5(+/- 2.5) X 10(7) M-1 s-1. Subsequent formation of a stable complex occurs with an apparent bimolecular rate constant kappa a = 6.7 X 10(6) M-1 s-1. At low total DNA concentration, the dissociation rate constant for the specific CAP-DNA complex is 1.2 X 10(-4) s-1. The ratio of formation and dissociation rate constants yields an estimate of the equilibrium constant, Keq = 5 X 10(10) M-1, in good agreement with static results. We observed that the dissociation rate constant of both CAP-DNA and repressor-DNA complexes is increased by adding non-specific "catalytic" DNA to the reaction mixture. CAP dissociation by the concentration-dependent pathway is second-order in added non-specific DNA, consistent with either the simultaneous or the sequential participation of two DNA molecules in the reaction mechanism. The results imply a role for distal DNA in assembly-disassembly of specific CAP-DNA complexes, and are consistent with a model in which the subunits in the CAP dimer separate in the assembly-disassembly process. The dissociation of lac repressor-operator complexes was found to be DNA concentration-dependent as well, although in contrast to CAP, the reaction is first-order in catalytic DNA. Added excess operator-rich DNA gave more rapid dissociation than equivalent concentrations of non-specific DNA, indicating that the sequence content of the competing DNA influences the rate of repressor dissociation. The simplest interpretation of these observations is that lac repressor can be transferred directly from one DNA molecule to another. A comparison of the translocation rates calculated for direct transfer with those predicted by the one-dimensional sliding model indicates that direct transfer may play a role in the binding site search of lac repressor.     

Comparison of the binding sites for the Escherichia coli cAMP receptor protein at the lactose and galactose promoters

Polyacrylamide gel electrophoresis has been used to visualise and quantitate complexes between the Escherichia coli cyclic AMP receptor protein (CRP) and DNA fragments containing the promoter region of either the E. coli galactose or lactose operons. We show that, although CRP binding to the gal fragment is weaker than binding to the lac fragment, in each case, stable complexes are formed between one dimer of CRP and one molecule of DNA. We have examined the effects of a series of deletions and point mutations in the gal promoter region on CRP binding. From the position of deletions and mutations which prevent the formation of stable complexes, we deduce the location and extent of the sequence at the CRP binding site. We show that it covers approximately the same length of sequence as the binding site at the lac promoter. Unlike the lac site, the gal site contains no palindromic sequence. We discuss the importance of symmetry in the sequence at CRP binding sites and the validity of CRP binding consensus sequences which have been proposed.     

Analysis by atomic force microscopy of Med8 binding to cis-acting regulatory elements of the SUC2 and HXK2 genes of saccharomyces cerevisiae.

Med8 protein is a regulator that specifically binds to upstream activating sequences (UASs) of SUC2 promoter, to downstream repressing sequences (DRSs) of the HXK2 gene and to the carboxy-terminal domain of the RNA polymerase II. Atomic force microscopy has allowed for direct visualization of Med8 interactions with a 305 bp fragment of SUC2 promoter and with a 676 bp fragment of HXK2 gene, containing respectively the UASs and DRSs regulatory regions. This approach has provided complementary information about the position and the structure of the DNA-protein complexes. Med8 binding to DNA results in total covering of one of the two existing 7 bp motives (consensus, (A/C)(A/G)GAAAT) in the studied DNA fragments. No preference for binding either of the two UASs of SUC2 promoter as well as for the two DRSs of HXK2 gene has been found. We also discuss whether this protein works as dimer or as a monomer.     

Regulation of open complex formation at the Escherichia coli galactose operon promoters. Simultaneous interaction of RNA polymerase, gal repressor and CAP/cAMP.

The regulation of open complex formation at the Escherichia coli galactose operon promoters by galactose repressor and catabolite activator protein/cyclic AMP (CAP/cAMP) was investigated in DNA-binding and kinetic experiments performed in vitro. We found that gal repressor and CAP/cAMP bind to the gal regulatory region independently, resulting in simultaneous occupancy of the two gal operators and the CAP/cAMP binding site. Both CAP/cAMP and gal repressor altered the partitioning of RNA polymerase between the two overlapping gal promoters. Open complexes formed in the absence of added regulatory proteins were partitioned between gal P1 and P2 with occupancies of 25% and 75%, respectively. CAP/cAMP caused open complexes to be formed nearly exclusively at P1 (98% occupancy). gal repressor caused a co-ordinated, but incomplete, switch in promoter partitioning from P1 to P2 in both the absence and presence of CAP/cAMP. We measured the kinetic constants governing open complex formation and decay at the gal promoters in the absence and presence of gal repressor and CAP/cAMP. CAP/cAMP had the largest effect on the kinetics of open complex formation, resulting in a 30-fold increase in the apparent binding constant. We conclude that the regulation of open complex formation at the gal promoters does not result from competition between gal repressor, CAP/cAMP and RNA polymerase for binding at the gal operon regulatory region, but instead results from the interactions of the three proteins during the formation of a nucleoprotein complex on the gal DNA fragment. Finally, we present a kinetic model for the regulation of open complex formation at the gal operon.     

Mutants of the lac promoter with large insertions and deletions between the CAP binding site and the -35 region

A set of the lac promoter mutants that have varying lengths of the spacer between the CAP binding site and the -35 region was constructed. The mutants have the spacer length increased by five (I5 mutant), or eleven (I11) residues or decreased by eleven residues (D11). We also present a construction of the hybrid between the gal and lac promoters in which the CAP binding site and the -35 region of the gal promoter are fused to the lac -10 region. The promoter fragments were assembled through ligations of chemically synthesized oligodeoxynucleotides and cloned into a pBR322-derivative vector. The results of the in vivo assays of promoter activity show that the I11 mutation results in an active but weak promoter that can be stimulated by CAP, though to a lesser degree than the wild-type lac promoter. The other mutants exhibit no promoter activity. Since the insertion of 11-bp preserves the location of the CAP binding site on the same side on the DNA helix, the data demonstrate the importance of spatial alignment between the CAP binding site and the promoter. The fact that the gal::lac hybrid is inactive as a promoter indicates also that catabolite activation is a highly complex process in which the -35 and -10 regions cannot be easily exchanged between promoters.