A role for TBP dimerization in preventing unregulated gene expression.

The recruitment of the TATA box-binding protein (TBP) to promoters in vivo is often rate limiting in gene expression. We present evidence that TBP negatively autoregulates its accessibility to promoter DNA in yeast through dimerization. The crystal structure of TBP dimers was used to design point mutations in the dimer interface. These mutants are impaired for dimerization in vitro, and in vivo they generate large increases in activator-independent gene expression. Overexpression of wild-type TBP suppresses these mutants, possibly by heterodimerizing with them. In addition to loss of autorepression, dimerization-defective TBPs are rapidly degraded in vivo. Direct detection of TBP dimers in vivo was achieved through chemical cross-linking. Taken together, the data suggest that TBP dimerization prevents unregulated gene expression and its own degradation.     

TFIIA regulates TBP and TFIID dimers.

Dimerization of the TATA-binding protein (TBP) through its DNA-binding domain blocks TBP from accessing DNA and prevents unregulated gene expression. TFIIA plays a central role in loading TBP and its multisubunit counterpart TFIID onto promoter DNA, and it is therefore a candidate for regulating TBP/TFIID dimerization. Here, we show that TFIIA promotes the dissociation of TBP dimers directly and in doing so accelerates the kinetics of DNA binding. TFIID dimer dissociation was found to be slow and rate limiting in DNA binding. TFIIA induced a rapid dissociation of TFIID dimers, allowing TFIID to readily load onto promoter DNA. Together, these results suggest a novel mechanism by which TFIIA assists in regulating gene expression.     

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.     

Influence of the N-terminal domain and divalent cations on self-association and DNA binding by the Saccharomyces cerevisiae TATA binding protein

The localization of a single tryptophan to the N-terminal domain and six tyrosines to the C-terminal domain of TBP allows intrinsic fluorescence to separately report on the structures and dynamics of the full-length TATA binding protein (TBP) of Saccharomyces cerevisiae and its C-terminal DNA binding domain (TBPc) as a function of self-association and DNA binding. TBPc is more compact than the C-terminal domain within the full-length protein. Quenching of the intrinsic fluorescence by DNA and external dynamic quenchers shows that the observed tyrosine fluorescence is due to the four residues surrounding the "DNA binding saddle" of the C-terminal domain. TBP's N-terminal domain unfolds and changes its position relative to the C-terminal domain upon DNA binding. It partially shields the DNA binding saddle in octameric TBP, shifting upon dissociation to monomers to expose the saddle to DNA. Structure-energetic correlations were obtained by comparing the contribution that electrostatic interactions make to DNA binding by TBP and TBPc; DNA binding by TBPc is more hydrophobic than that by TBP, suggesting that the N-terminal domain either interacts with bound DNA directly or screens a part of the C-terminal domain, diminishing its electronegativity. The competition between divalent cations, K+, and DNA is not straightforward. Divalent cations strengthen binding of TBP to DNA and do so more strongly for TBPc. We suggest that divalent cations affect the structure of the bound DNA perhaps by stabilizing its distorted conformation in complexes with TBPc and TBP and that the N-terminal domain mimics the effects of divalent cations. These data support an autoinhibitory mechanism in which competition between the N-terminal domain and DNA for the saddle diminishes the DNA binding affinity of the full-length protein.     

DNA-induced dimerization of the Escherichia coli rep helicase. Allosteric effects of single-stranded and duplex DNA.

The Escherichia coli Rep helicase is a stable monomer (Mr = 72,802) in the absence of DNA; however, binding of single-stranded (ss) or duplex (ds) DNA induces Rep monomers to dimerize. Furthermore, a chemically cross-linked Rep dimer retains both its DNA-dependent ATPase and helicase activities, suggesting that the functionally active Rep helicase is a dimer (Chao, K., and Lohman, T. M. (1991) J. Mol. Biol. 221, 1165-1181). Using a modified "double-filter" nitrocellulose filter binding assay, we have examined quantitatively the equilibrium binding of Rep to a series of ss-oligodeoxynucleotides, d(pN)n (8 less than or equal to n less than or equal to 20) and two 16-base pair duplex oligodeoxynucleotides, which are short enough so that only a single Rep monomer can bind to each oligonucleotide. This strategy has enabled us to examine the linkage between DNA binding and dimerization. We also present a statistical thermodynamic model to describe the DNA-induced Rep dimerization in the presence of ss- and/or ds-oligodeoxynucleotides. We observe quantitative agreement between this model and the experimental binding isotherms and have analyzed these isotherms to obtain the seven independent interaction constants that describe Rep-DNA binding and Rep dimerization. We find that Rep monomers (P) can bind either ss-DNA (S) or ds-DNA (D) to form PS or PD, respectively, which can then dimerize to form P2S or P2D. Furthermore, both protomers of the DNA-induced Rep dimer can bind DNA to form either P2S2, P2D2 or the mixed dimer species P2SD and ss- and ds-DNA compete for the same sites on the Rep protein. When bound to DNA, the Rep dimerization constants are approximately 1-2 x 10(8) M-1 (6 mM NaCl, pH 7.5, 4 degrees C), which are greater than the dimerization constant for free Rep monomers by at least 10(4)-fold. The Rep-ss-DNA interaction constants are independent of base composition and sequence, consistent with its role as a nonspecific DNA-binding protein. Allosteric effects are associated with ss- and ds-DNA binding to the half-saturated Rep dimers, i.e. the affinity of either ss- or ds-DNA to the free promoter of a half-saturated Rep dimer is clearly influenced by the conformation of DNA bound to the first protomer. These allosteric effects further support the proposal that the Rep dimer is functionally important and that the Rep-DNA species P2S2 and P2SD may serve as useful models for intermediates that occur during DNA unwinding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of the non-conserved N-terminus on the DNA binding activity of the yeast TATA binding protein.

We have studied the DNA binding activity of recombinant yeast TATA Binding Protein (TBP) with particular interest in the role played by the non-conserved N-terminal domain. By comparing the DNA binding activity of wild type yeast TBP with a mutant form of TBP that lacks the non-conserved N-terminal domain (TBP delta 57), we have determined that the N-terminus of TBP alters both the shape and the stability of the TBP-DNA complex. Measurements of the DNA bending angle indicate that the N-terminus enhances the bending of the DNA that is induced by TBP binding and greatly destabilizes the TBP-DNA complex during native gel electrophoresis. In solution, the N-terminus has only a slight effect on the equilibrium dissociation constant and the dissociation rate constant. However, the N-terminal domain reduces the association rate constant in a temperature dependent manner and increases the apparent activation energy of the TBP-DNA complex formation by 3 kcal/mole. These data suggest that a conformational change involving the N-terminus of TBP may be one of the isomerization steps in the formation of a stable TBP-DNA complex.     

The amino-terminal tails of the core histones and the translational position of the TATA box determine TBP/TFIIA association with nucleosomal DNA.

We establish that the TATA binding protein (TBP) in the presence of TFIIA recognizes the TATA box in nucleosomal DNA dependent on the dissociation of the amino-terminal tails of the core histones from the nucleosome and the position of the TATA box within the nucleosome. We examine TBP/TFIIA access to the TATA box with this sequence placed in four distinct rotational frames with reference to the histone surface and at three distinct translational positions at the edge, side and dyad axis of the nucleosome. Under our experimental conditions, we find that the preferential translational position at which TBP/TFIIA can bind the TATA box is within linker DNA at the edge of the nucleosome and that binding is facilitated if contacts made by the amino-terminal tails of the histones with nucleosomal DNA are eliminated. TBP/TFIIA binding to DNA at the edge of the nucleosome occurs with the TATA box in all four rotational positions. This is indicative of TBP/TFIIA association directing the dissociation of the TATA box from the surface of the histone octamer.     

Ordered disruption of subunit interfaces during the stepwise reversible dissociation of Escherichia coli phosphofructokinase with KSCN

The reversible inactivation and dissociation of the allosteric phosphofructokinase from Escherichia coli has been studied in relatively mild conditions, i.e., in the presence of the chaotropic agent KSCN. At moderate KSCN concentration, the loss of enzymatic activity involves two separated phases: first, a rapid dissociation of part of the tetramer into dimers, second, a slower displacement of the dimer-tetramer equilibrium upon further dissociation of the dimer into monomers. These two reactions can no longer be distinguished above 0.3 M KSCN since complete inactivation occurs in a single reaction. Different changes are observed for the fluorescence and the activity of the enzyme in KSCN: the fluorescence is not affected by the dissociation into dimers which is responsible for inactivation. The decrease in fluorescence reflects the change in environment of the unique tryptophan residue, Trp 311, during the dimer to monomer dissociation. This residue belongs to the interface containing the regulatory site, and its native fluorescence indicates that this interface is still present in the dimer. The substrate fructose 6-phosphate protects phosphofructokinase from inactivation by binding to the tetramer and prevents its dissociation into dimers. The presence of phosphoenolpyruvate prevents the slow dissociation of the dimer into monomers, which shows the ability of the dimer to bind the inhibitor. Two successive processes can be observed during reassociation of the protein upon KSCN dilution. First, a fast reaction (k1 = 2 x 10(5) M-1.s-1) is accompanied by a fluorescence increase and results in the formation of the dimeric species.(ABSTRACT TRUNCATED AT 250 WORDS)