Role of BvgA phosphorylation and DNA binding affinity in control of Bvg-mediated phenotypic phase transition in Bordetella pertussis

To investigate the mechanism by which the Bordetella BvgAS phosphorelay controls expression of at least three distinct phenotypic phases, we isolated and characterized two B. pertussis mutants that were able to express Bvg- and Bvg(i) phase phenotypes but not Bvg+ phase phenotypes. In both cases, the mutant phenotype was due to a single nucleotide change in bvgA resulting in a single amino acid substitution in BvgA. In vitro phosphorylation assays showed that BvgA containing the T194M substitution was significantly impaired in its ability to use either BvgS or acetyl phosphate as a substrate for phosphorylation. Binding studies indicated that this mutant protein was able to bind an oligonucleotide containing a high-affinity BvgA binding site in a manner similar to wild-type BvgA, but was defective for binding the fhaB promoter in the absence of RNA polymerase (RNAP). By contrast, BvgA containing the R152H substitution had wild-type phosphorylation properties but was severely defective in its ability to bind either the high-affinity BvgA binding site-containing oligonucleotide or the fhaB promoter by itself. Both mutant BvgA proteins were able to bind the fhaB promoter in the presence of RNAP however, demonstrating the profound effect that RNAP has on stabilizing the ternary complexes between promoter DNA, BvgA and RNAP. Our results are consistent with the hypothesis that BvgAS controls expression of multiple phenotypic phases by adjusting the intracellular concentration of BvgA-P and they demonstrate the additive nature of BvgA binding site affinity and protein-protein interactions at different Bvg-regulated promoters.     

Binding modes of the initiator and inhibitor forms of the replication protein pi to the gamma ori iteron of plasmid R6K

Discerning the interactions between initiator protein and the origin of replication should provide insights into the mechanism of DNA replication initiation. In the gamma origin of plasmid R6K, the Rep protein, pi, is distinctive in that it can bind the seven 22-bp iterons in two forms; pi monomers activate replication, whereas pi dimers act as inhibitors. In this work, we used wild type and variants of the pi protein with altered monomer/dimer ratios to study iteron/pi interactions. High resolution contact mapping was conducted using multiple techniques (missing base contact probing, methylation protection, base modification, and hydroxyl radical footprinting), and the electrophoretic separation of nucleoprotein complexes allowed us to discriminate between contact patterns produced by pi monomers and dimers. We also isolated iteron mutants that affected the binding of pi monomers (only) or both monomers and dimers. The mutational studies and footprinting analyses revealed that, when binding DNA, pi monomers interact with nucleotides spanning the entire length of the iteron. In contrast, pi dimers interact with only the left half of the iteron; however, the retained interactions are strikingly similar to those seen with monomers. These results support a model in which Rep protein dimerization disturbs one of two DNA binding domains important for monomer/iteron interaction; the dimer/iteron interaction utilizes only one DNA binding domain.     

Inhibition of TATA binding protein dimerization by RNA polymerase III transcription initiation factor Brf1

The Brf1 subunit of TFIIIB plays an important role in recruiting the TATA-binding protein (TBP) to the up-stream region of genes transcribed by RNA polymerase III. When TBP is not bound to promoters, it sequesters its DNA binding domain through dimerization. Promoter assembly factors therefore might be required to dissociate TBP into productively binding monomers. Here we show that Saccharomyces cerevisiae Brf1 induces TBP dimers to dissociate. The high affinity TBP binding domain of Brf1 is not sufficient to promote TBP dimer dissociation but in addition requires the TFIIB homology domain of Brf1. A model is proposed to explain how two distinct functional domains of Brf1 work in concert to dissociate TBP into monomers.     

Determinants of high-affinity DNA binding by the glucocorticoid receptor: evaluation of receptor domains outside the DNA-binding domain.

In this study, we have investigated the influence of regions outside the DNA-binding domain of the human glucocorticoid receptor on high-affinity DNA binding. We find that the DNA-binding domain shows a 10-fold lower affinity for a palindromic DNA-binding site than the intact receptor. The N-terminal part of the receptor protein does not influence its DNA-binding affinity, while the C-terminal steroid-binding domain increases the DNA-binding affinity of the receptor molecule. It has previously been shown that both the intact glucocorticoid receptor and the glucocorticoid receptor DNA-binding domain bind to a palindromic glucocorticoid response element on DNA as dimers. It is likely that differences in DNA-binding affinity observed result from protein-protein interactions outside the DNA-binding domain between receptor monomers, as has been shown for the estrogen receptor. We have previously identified a segment involved in protein-protein interactions between DNA-binding domains of glucocorticoid receptors. This, in combination with results presented in this study, suggests that there are at least two sites of contact between receptor monomers bound to DNA. We suggest that the interaction between the DNA-binding domains may act primarily to restrict DNA binding to binding sites with appropriate half-site spacing and that additional stability of the receptor dimer is provided by the interactions between the steroid-binding domains.     

Dimerization of signalling modules of the EvgAS and BvgAS phosphorelay systems

Biophysical and biochemical properties of signalling proteins or domains derived from the unorthodox EvgAS and BvgAS two-component phosphorelay systems of Escherichia coli and Bordetella pertussis were investigated. Oligomerization of the effector proteins EvgA and BvgA and of truncated EvgS and BvgS derived signalling proteins containing the receiver and histidine containing phosphotransfer (HPt) domains or comprising only the HPt domains were characterized by native gel electrophoresis, gel permeation experiments and analytical ultracentrifugation. The results obtained by the different methods are consistent with non-phosphorylated EvgA and BvgA proteins being dimers in solution with a dissociation constant significantly below 1 microM. In contrast, all sensor derived domains of EvgS and BvgS were observed to be monomers in vitro. No indications for a phosphorylation induced stimulation of oligomerization of the C-terminal histidine kinase domains could be detected. In agreement with these data, surface plasmon resonance studies revealed a 2:1 stoichiometry in the interaction of EvgA with the immobilized EvgS HPt domain and an affinity constant of 1. 24x10(6) M(-1).     

Multimerization of the Drosophila zeste protein is required for efficient DNA binding.

The Drosophila zeste protein forms multimeric species in vitro through its C-terminal domain. Multimerization is required for efficient binding to DNA containing multiple recognition sequences and increasing the number of binding sites stimulates binding in a cooperative manner. Mutants that can only form dimers still bind to a dimeric site, but with lower affinity. Mutations or progressive deletions from the C-terminal show that when even dimer formation is prevented, DNA-binding activity is lost. Surprisingly, binding activity is regained with larger deletions that leave only the DNA-binding domain. Additional protein sequences apparently inhibit DNA binding unless they permit multimerization. The DNA-binding domain peptides bind strongly even to isolated recognition sequences and they bind as monomers. The ability of various zeste peptides to stimulate white gene expression in vivo shows that multimeric forms are the functional species of the zeste product in vivo. The DNA-binding domain peptide binds well to DNA in vitro, but it cannot stimulate white gene expression in vivo. This failure may reflect the need for an activation domain or it may be caused by indiscriminate binding of this peptide to non-functional isolated sites. Multimerization increases binding specificity, selecting only sites with multiple recognition sequences.     

Study of the interaction of DNA and acridine orange by various optical methods

The degree of binding of acridine orange to DNA, native or denatured, has been determined by equilibrium dialysis in 0.1M and 0.001M NaCl at 20°. The nature of the binding process has been investigated by studying various optical properties of the dye–DNA complexes and by relating them to the binding ratio. All these properties were found to vary quantitatively and qualitatively according to the successive stages of the process. These stages were assumed to be a strong binding of intercalated monomers followed by formation of bound dimers and finally by external binding of aggregates of native DNA. Absorption spectra of the complexes could be interpreted on that basis. Circular dichroism spectra were resolved into components: one band for intercalated monomers without interactions, two excition splittings for interacting monomers and bound dimers, respectively, weak bands and exciton splitting for external aggregates. The fluorescence intensity was greatly enhanced in intercallated monomers; its quenching at higher binding ratio was quantitatively related to dimer fixation. The value of the anisotropy of fluorescence at low binding ratio suggested a limited mobility of intercalated monomers; the decrease of polarization at higher binding was attributed to energy transfer between monomers. Electric dichroism displayed by the complexes in the dye absorption bands indicated an orientation of the bound molecules quite parallel to the base rings at low binding. In the range of fixation of dimers and external molecules, the dichroism was lower but still indicated an important degree of ordering.