Selective binding of the TATA box-binding protein to the TATA box-containing promoter: analysis of structural and energetic factors

Structural comparisons have led to the suggestion that the conformational rearrangement that would be required to change A-DNA into the TA-DNA form of DNA observed in the complex with the TATA box binding protein (TBP) could be completed by modifying only the value of the glycosyl bond chi by approximately 45 degrees. The lack of a high number of crystal structures of this type makes it difficult to conclude whether a smooth transition from A-DNA to TA-DNA can occur without disrupting at any point either the Watson-Crick base pairing or the A-DNA conformation of the backbone. To explore the possibility of such a smooth transition, constrained molecular dynamics simulations were carried out for the double-stranded dodecamer d(GGTATATAAAAC), in which a transition from A-DNA to TA-DNA was induced by modifying only the chi angle values. The results demonstrate the feasibility of a continuous path in the A-DNA to TA-DNA transition. Varying extents of DNA curvature are also attainable, by maintaining the A-DNA backbone structure and Watson-Crick hydrogen bonding while changing the chi angle value smoothly from that in A-DNA to one corresponding to B-DNA.     

Studies of the interaction between Rad52 protein and the yeast single-stranded DNA binding protein RPA

The RFA1 gene encodes the large subunit of the yeast trimeric single-stranded DNA binding protein replication protein A (RPA), which is known to play a critical role in DNA replication. A Saccharomyces cerevisiae strain carrying the rfa1-44 allele displays a number of impaired recombination and repair phenotypes, all of which are suppressible by overexpression of RAD52. We demonstrate that a rad52 mutation is epistatic to the rfa1-44 mutation, placing RFA1 and RAD52 in the same genetic pathway. Furthermore, two-hybrid analysis indicates the existence of interactions between Rad52 and all three subunits of RPA. The nature of this Rad52-RPA interaction was further explored by using two different mutant alleles of rad52. Both mutations lie in the amino terminus of Rad52, a region previously defined as being responsible for its DNA binding ability (U. H. Mortenson, C. Beudixen, I. Sunjeuaric, and R. Rothstein, Proc. Natl. Acad. Sci. USA 93:10729-10734, 1996). The yeast two-hybrid system was used to monitor the protein-protein interactions of the mutant Rad52 proteins. Both of the mutant proteins are capable of self-interaction but are unable to interact with Rad51. The mutant proteins also lack the ability to interact with the large subunit of RPA, Rfa1. Interestingly, they retain their ability to interact with the medium-sized subunit, Rfa2. Given the location of the mutations in the DNA binding domain of Rad52, a model incorporating the role of DNA in the protein-protein interactions involved in the repair of DNA double-strand breaks is presented.     

Mammalian protein RAP46: an interaction partner and modulator of 70 kDa heat shock proteins

A ubiquitously expressed nuclear receptor-associating protein of approximately 46 kDa (RAP46) was identified recently. Interaction experiments with in vitro-translated proteins and proteins contained in cell extracts revealed that a great variety of cellular regulators associate with RAP46. However, in direct interaction tests by the far-Western technique, only 70 kDa proteins showed up and were identified as members of the 70 kDa heat shock protein (hsp70) family. Interaction is specific since not all members of the hsp70 family bind to RAP46; interaction occurs through their ATP-binding domain. RAP46 forms complexes with hsp70 in mammalian cells and interacts with hsp70 in the yeast two-hybrid system. Consistent with the fact that hsp70 can bind a multitude of proteins, we identified heteromeric complexes of RAP46-hsp70 with some selected proteins, most notably c-Jun. Complex formation is increased significantly by pre-treatment with alkaline phosphatase, thus suggesting modulation of interactions by protein phosphorylation. We observed that RAP46 interferes with efficient refolding of thermally denatured luciferase. Moreover, ATP-dependent binding of misfolded proteins to hsp70 was greatly inhibited by RAP46. These data suggest that RAP46 functions as a regulator of hsp70 in higher eukaryotes.     

Analysis of co-crystal structures to identify the stereochemical determinants of the orientation of TBP on the TATA box

Possible stereochemical determinants of the orientation of TBP on the TATA box are discussed using the crystal coordinates of TBP-TATA complexes, which have been determined by other groups. The C-terminal half of the TBP beta-sheet interacts with the TATA site of the DNA, and the N-terminal half with the A-rich site, so that the two sites with distinct curvatures produce a unique fit. Although chemical contacts take place between one side of the beta-sheet and the DNA minor groove, the interaction seems to be facilitated indirectly by the characteristics of the other side of the beta-sheet and the DNA major groove. Thus, Ala71, Leu162 and Pro190 differentiate the curvature of the beta-sheet in the N- and C-halves. The methyl positions in the DNA major groove modulate the bendability of the two DNA sites by using differences in the rolling capacity of TA and AT compared with PyT, and in the shifting capacity of AT compared with TT. The deformations of the first steps (TA and PyT) in the two sites are the largest and thus are important for the overall bending of the DNA. The differences between the two DNA sites are greatest at the second steps (AT and TT) and so these are important for determining the orientation of TBP.     

Overproduced and purified receptor binding protein pb5 of bacteriophage T5 binds to the T5 receptor protein FhuA

A promotor-less oad gene of bacteriophage T5, encoding the receptor binding protein pb5, was cloned into pT7-3 under the control of phage T7 promoter phi 10. Induction with IPTG resulted in enhanced production of pb5. Upon fractionation of the producing cells, most of the overproduced pb5 was found in the membrane fraction, which was most likely due to aggregation of the protein. The minor, soluble fraction of pb5 specifically inhibited adsorption of T5 to its FhuA receptor protein. Inhibition was also seen with trace amounts of pb5, and binding of pb5 to FhuA appeared to be almost irreversible. Purification of pb5 from the cytosolic fraction was performed by FPLC using a MonoQ column. pb5, which did not bind to the matrix of the column, was obtained in almost pure form. The purified protein also inhibited T5 adsorption.     

DNA sequence-specific recognition by the Saccharomyces cerevisiae "TATA" binding protein: promoter-dependent differences in the thermodynamics and kinetics of binding

The equilibrium binding and association kinetics of the Saccharomyces cerevisiae TATA Binding Protein (TBP) to the E4 and Major Late promoters of adenovirus (TATATATA and TATAAAAG, respectively), have been directly compared by quantitative DNase I titration and quench-flow "footprinting". The equilibrium binding of TBP to both promoters is described by the equilibrium TBP + DNA"TATA" left and right arrow TBP-DNA"TATA". The salt dependence of TBP binding to both promoters is identical within experimental error while the temperature dependence differs significantly. The observed rate of association follows simple second-order kinetics over the TBP concentration ranges investigated. The salt and temperature dependencies of the second-order association rate constants for TBP binding the two promoters reflect the dependencies determined by equilibrium binding. The TBP-E4 promoter interaction is entropically driven at low temperature and enthalpically driven at high temperature while the TBP-Major Late promoter reaction is entropically driven over virtually the entire temperature range investigated. These data suggest that the reaction mechanisms of TBP-promoter interactions are TATA sequence-specific and provide for differential regulation of promoters as a function of environmental variables.     

Characterization of the binding properties of protein LG, an immunoglobulin-binding hybrid protein

Protein LG is a 50-kDa hybrid molecule containing four Ig-light-chain-binding domains from protein L of Peptostreptococcus magnus and two IgG-Fe-binding repeats from streptococcal protein G. Here we analyse the binding of protein LG to Ig from several mammalian species. Protein LG was shown to bind human IgG of all subclasses and other Ig classes that carry kappa chains. The binding to human IgG was only marginally influenced by changes in temperature (4-37 degrees C) or salt concentration (0-1.6 M), and was stable over a wide pH range (pH 4-10). Protein LG bound to Ig from 11 of 12 mammalian species, including those of rabbit, mouse and rat. The affinity constants obtained for the interactions between protein LG and polyclonal IgG from rabbit (4.0 x 10(9) M-1), mouse (1.7 x 10(9) M-1) and rat (1.3 x 10(9) M-1) were similar to the value previously reported for the interaction between the hybrid protein and human polyclonal IgG (5.9 x 10(9) M-1). The interaction between protein LG and a mouse IgG mAb was not influenced by the presence of the specific protein antigen, nor was the binding of this antibody to its ligand affected by protein LG. Inhibition experiments demonstrated that the Ig-binding site of one of the fusion partners retained its ligand-binding capacity when the other component was occupied. Protein LG selectively absorbed 85-90% of the total Ig present in human and rabbit sera and 75-80% of the Ig in sera from mouse and rat. Human serum depleted of C1q, factor D and properdin and preabsorbed by protein LG could be used as a source for other complement factors. These data demonstrate that protein LG is a very versatile Ig-binding protein.     

Multiple binding sites in the interaction between an extracellular fibrinogen-binding protein from Staphylococcus aureus and fibrinogen

Efb (previously Fib) is a fibrinogen-binding protein secreted by Staphylococcus aureus. It has previously been shown that it plays a role in a wound infection model in the rat and that antibodies against Efb reduce the number of recovered bacteria from the mammary glands in a mouse mastitis model. Efb binds to the alpha-chain of fibrinogen and does not participate in bacterial adherence to fibrinogen. The binding of Efb to fibrinogen is divalent, with one binding site within the two repeat regions in Efb at the N terminus and one binding site at the C terminus. The divalent binding nature leads to precipitation of Efb-fibrinogen complex when the proteins are added to each other at a 1:1 molar ratio. The interaction between Efb and fibrinogen is strongly enhanced by Ca2+ or Zn2+ but not by Mg2.     

Childhood hepatoblastomas frequently carry a mutated degradation targeting box of the beta-catenin gene

The enzyme system responsible for the N-deacetylation of eprinomectin in rats was characterized. Tissue and subcellular studies showed that the hydrolysis activity was localized mainly in liver microsomes. Apparent KM and Vmax values calculated from Lineweaver-Burk plots were 53 microM and 0.81 nmol/mg/min for male rats and 70 microM and 4.99 nmol/mg/min for female rats, respectively. Pretreatment of male rats with dexamethasone, phenobarbital, and pregnenolone 16alpha-carbonitrile increased the activity by more than 3-fold. Paraoxon and bis-4-nitrophenylphosphate strongly inhibited the deacetylase activity at concentrations as low as 1 microM. The hydrolysis activity also was inhibited by SKF525, but less effectively. Eserine strongly inhibited the activity at 1 x 10(-4) M. HgCl2 decreased the activity to about 40% at a concentration of 1 x 10(-4) M. FeCl3, CaCl2, MgCl2, and EDTA had little effect on the hydrolysis of eprinomectin, whereas NaF slightly increased the activity to 118%. Thus, the inhibition study suggested that eprinomectin deacetylase resembled "B" type carboxylesterase/amidases. The hydrolysis activity of eprinomectin and isocarboxazid, a specific substrate of RL2 [Hosokawa, M, Maki T and Satoh T (1987) Mol Pharmacol 31:579-584], by liver microsomes from rats treated with various cytochrome P-450 inducers correlated well (r = 0.92). Also, elusion profiles of esterase by gel filtration and ion exchange chromatography demonstrated that the active protein(s) for eprinomectin and isocarboxazid hydrolysis coeluted. Thus, RL2 or an enzyme system similar to RL2 is responsible for the N-deacetylation of eprinomectin.