Wild-type Tetramer Research Articles

How p53 binds DNA as a tetramer.

The p53 tumor suppressor protein is a tetramer that binds sequence-specifically to a DNA consensus sequence consisting of two consecutive half-sites, with each half-site being formed by two head-to-head quarter-sites (--><-- --><--). Each p53 subunit binds to one quarter-site, resulting in all four DNA quarter-sites being occupied by one p53 tetramer. The tetramerization domain forms a symmetric dimer of dimers, and two contrasting models have the two DNA-binding domains of each dimer bound to either consecutive or alternating quarter-sites. We show here that the two monomers within a dimer bind to a half-site (two consecutive quarter-sites), but not to separated (alternating) quarter-sites. Tetramers bind similarly, with the two dimers within each tetramer binding to pairs of half-sites. Although one dimer within the tetramer is sufficient for binding to one half-site in DNA, concurrent interaction of the second dimer with a second half-site in DNA drastically enhances binding affinity (at least 50-fold). This cooperative dimer-dimer interaction occurs independently of tetramerization and is a primary mechanism responsible for the stabilization of p53 DNA binding. Based on these findings, we present a model of p53 binding to the consensus sequence, with the tetramer binding DNA as a pair of clamps.

A natural variant of bovine dopamine β-monooxygenase with phenylalanine as residue 208: purification and characterization of the variant homo- and heterotetramers of (F208) 4 and (F208) 2(L208) 2

Bovine dopamine β-monooxygenase was purified from each of 18 individual adrenal glands by the method we have developed for the rapid purification of the enzyme from a single adrenal gland. Differential peptide mapping of the 18 enzyme preparations following fluorescence labeling of their cysteine residues revealed the presence of a novel variant with Phe as residue 208 in 14 adrenal glands; seven of them were homozygous for the variant allele and the remaining seven heterozygous. The variant enzyme was a tetramer and exhibited kinetic and structural properties similar to those of the wild-type tetramer (L208) 4. These results indicate an allelic polymorphism and codominant expression of the two alleles of the enzyme gene.

P53 oligomerization and DNA looping are linked with transcriptional activation.

We examined the role of p53 oligomerization in DNA binding and in transactivation. By conventional electron microscopy (EM) and scanning transmission EM, we find that wild-type tetramers contact 18-20 bp at single or tandem 19 bp consensus sequences and also stack in apparent register, tetramer on top of tetramer. Stacked tetramers link separated DNA binding sites with DNA loops. Interestingly, the p53(1-320) segment, which lacks the C-terminal tetramerization domain, binds DNA consensus sites as stacked oligomers. Although the truncated protein binds DNA with reduced efficiency, it nevertheless induces DNA looping by self-association. p53, therefore, has a C-terminal tetramerization domain that enhances DNA binding and a non-tetrameric oligomerization domain that stacks p53 at consensus sites and loops separated consensus sites via protein-protein interactions. Using model promoters, we demonstrate that wild-type and tetramerization-deficient p53s activate transcription well when tandem consensus sites are proximal to TATA sequences and poorly when tandem sites are distal. In the presence of proximal sites, however, stimulation by distal sites increases 25-fold. Tetramerization and stacking of tetramers, therefore, provide dual mechanisms to augment the number of p53 molecules available for activation through p53 response elements. DNA looping between separated response elements further increases the concentration of local p53 by translocating distally bound protein to the promoter.

Origin of the Asymmetrical Contact between lac Repressor and lac Operator DNA

The Escherichia coli lac operator DNA contains two sequence repeats related by a pseudo-dyad axis. Deviations from symmetry, in the central 21 bp sequence, occur at two pairs of symmetrically related sites (+15/+7, +13/+9) and at the central base-pair +11. Mutational analysis and DNA protection studies have suggested asymmetric interactions of lac repressor along this sequence. Previous biophysical studies on the lac repressor-operator system have typically employed symmetrized operator sequences to simplify analysis. As a result, it has remained difficult to assess the importance of the naturally occurring sequence deviations from symmetry. Here, 19F-NMR is used to determine if the wild-type E. coli lac operator DNA sequence itself specifies a pair of distinct half-site interactions with lac repressor DNA binding domains. To observe protein interactions simultaneously at operator half-sites using 19 F-NMR, three pairs of naturally occurring, symmetry related thymine residues (at +6/+16, +8/+14 and +1/+21) were substituted pair wise by 5-fluoro-deoxyuridines (5-FdU). Two polypeptides corresponding to the N-terminal DNA binding domain of lac repressor "headpiece", residues 1 to 56 and 1 to 64, were employed to remove the steric constraints of subunit interaction in the wild-type tetramer. Spectral changes associated with headpiece binding to left side DNA sequences differ from those caused by binding to equivalent sequences on the right half-site. These results are similar to non-symmetric intact tetramer repressor interactions specified by the DNA sequence. Three mutant lac operator sequences with increased symmetry, bearing FdU substitutions were used to identify the relative importance of the three naturally asymmetric positions. Symmetrizing one pair of these sites alone or in addition to removing the central base-pair failed to produce identical NMR signal changes characteristic of symmetric headpiece—DNA complexes. However, symmetrizing both asymmetric pairs gave chemical shift changes expected from symmetric protein—DNA complexes. We propose that key interactions with the left side +9 (G · C) are altered at the symmetrically related right side +13 (A · T). The data show that the DNA sequence at +13 influences interactions three base-pairs away.

Site-directed mutagenesis of human protein disulphide isomerase: effect on the assembly, activity and endoplasmic reticulum retention of human prolyl 4-hydroxylase in Spodoptera frugiperda insect cells.

Protein disulphide isomerase (PDI) is a highly unusual multifunctional polypeptide, identical to the beta-subunit of prolyl 4-hydroxylase. It has two -Cys-Gly-His-Cys- sequences which represent two independently acting catalytic sites of PDI activity. We report here on the expression in baculovirus vectors of various mutant PDI/beta-subunits together with a wild-type alpha-subunit of the human prolyl 4-hydroxylase alpha 2 beta 2 tetramer in Spodoptera frugiperda insect cells. When either one or both of the -Cys-Gly-His-Cys- sequences was converted to -Ser-Gly-His-Cys-, a tetramer was formed as with wild-type PDI/beta-subunit. This tetramer was fully active prolyl 4-hydroxylase. The data demonstrate that PDI activity of the PDI/beta-subunit is not required for tetramer assembly or for the prolyl 4-hydroxylase activity of the tetramer, and thus other sequences of the PDI/beta-subunit may be critical for keeping the alpha-subunits in a catalytically active, non-aggregated conformation. Measurements of the PDI activities of tetramers containing the various mutant PDI/beta-subunits demonstrated that the activity of the wild-type tetramer is almost exclusively due to the C-terminal PDI catalytic sites, which explains the finding that the PDI activity of the PDI/beta-subunit present in the tetramer is about half that in the free polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Monomers of the Escherichia coli SSB-1 mutant protein bind single-stranded DNA

The Escherichia coli wild-type single strand binding (SSB) protein is a stable tetramer that binds to single-stranded (ss) DNA in its role in DNA replication, recombination and repair. The ssb-1 mutation, a substitution of tyrosine for histidine-55 within the SSB-1 protein, destabilizes the tetramer with respect to monomers, resulting in a temperature-sensitive defect in a variety of DNA metabolic processes, including replication. Using quenching of the intrinsic SSB-1 tryptophan fluorescence, we have examined the equilibrium binding of the oligonucleotide, dT(pT) 15, to the SSB-1 protein in order to determine whether a ssDNA binding site exists within individual SSB-1 monomers or whether the formation of the SSB tetramer is necessary for ssDNA binding. At high SSB-1 protein concentrations, such that the tetramer is stable, we find that four molecules of dT(pT) 15 bind per tetramer in a manner similar to that observed for the wild-type SSB tetramer; i.e. negative co-operativity is observed for ssDNA binding to the SSB-1 protomers. As a consequence of this negative co-operativity, binding is biphasic, with two molecules of dT(pT) 15 binding to the tetramer in each phase. However, the intrinsic binding constant, K 16, for the SSB-1 protomer-dT(pT) 15 interaction is a factor of 3 lower than for the wild-type protomer interaction and the negative co-operativity parameter, σ 16, is larger in the case of the SSB-1 tetramer, indicating a lower degree of negative co-operativity. At lower SSB-1 concentrations, SSB-1 monomers bind dT(pT) 15 without negative co-operativity; however, the intrinsic affinity of dT(pT) 15 for the monomer is a factor of ~10 lower than for the protomer (50 m m-NaCl, pH 8.1, 25 °C). Therefore, an individual SSB-1 monomer does possess an independent ssDNA binding site; hence formation of the tetramer is not required for ssDNA binding, although tetramer formation does increase the binding affinity significantly. These data also show that the negative co-operativity among ssDNA binding sites within an SSB tetramer is an intrinsic property of the tetramer. On the basis of these studies, we discuss a modified explanation for the temperature-sensitivity of the ssb-1 phenotype.

DNA looping in cellular repression of transcription of the galactose operon.

Communication between distant DNA sites is a central feature of many DNA transactions. Negative regulation of the galactose (gal) operon of Escherichia coli requires repressor binding to two operator sites located on opposite sides of the promoter. The proposed mechanism for regulation involves binding of the repressor to both operator sites, followed by a protein-protein association that loops the intervening promoter DNA (double occupancy plus association). To assess these requirements in vivo, we have previously converted gal operator sites to lac and shown that both operator sites must be occupied by the homologous repressor protein (Lac or Gal) for negative regulation of the gal operon. We have now addressed more directly the need for protein-protein association by the use of the converted operator sites and a mutant Lac repressor defective in association of the DNA-binding dimers. We have compared the biological and biochemical activity of two Lac repressors: the wild-type (tetramer) I+ form, in which the DNA-binding dimer units are tightly associated; and the mutant Iadi repressor, in which the dimer units do not associate effectively. The I+ repressor is an efficient negative regulator of the gal operon in vivo, but the Iadi mutant is an ineffective repressor. Purified I+ repressor efficiently forms DNA loops between operator sites that we have visualized by electron microscopy; the Iadi repressor fails to form DNA loops, although the protein binds effectively to both operator sites. From the clear correlation between looping in vitro and repression in vivo, we conclude that regulation of the gal operon depends on the association of repressor proteins bound to the two operator sites.(ABSTRACT TRUNCATED AT 250 WORDS)