DNA triple helix stabilisation by a naphthylquinoline dimer.
(33/1876)
We have used DNase I footprinting to examine the effect of a novel naphthylquinoline dimer, designed as a triplex-specific bis-intercalator, on the stability of intermolecular DNA triplexes. We find that this compound efficiently promotes triplex formation between the 9-mer oligonucleotide 5'-TTTTTTCTT and its oligopurine duplex target at concentrations as low as 0.1 microM, enhancing the triplex stability by at least 1000-fold. This compound, which is the first reported example of a triplex bis-intercalator, is about 30 times more potent than the simple monofunctional ligand. (+info)
Structural characterization of DNA and RNA sequences recognized by the gene 5 protein of bacteriophage fd.
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The single-stranded DNA sequence d(GT5G4CT4C) occurs close to the origin of replication within the intergenic region of the viral strand of bacteriophage fd. The RNA analogue of this sequence r(GU5G4CU4C) forms part of the untranslated leader sequence of the gene 2 mRNA and is specifically bound by the fd gene 5 protein in its role as a translational repressor. The structure of these sequences is likely to have an important role in the control of both DNA replication and RNA translation in the phage. We show that this 16 nt sequence, in both a DNA and an RNA context, can exist in a structured and unstructured form as determined by high-resolution gel filtration and non-denaturing gel electrophoresis. The CD spectrum of the structured form is characteristic of parallel guanine tetraplexes. The structured form of the DNA sequence melts at approx. 47 degrees C in the presence of Na+ ions but the structure is stabilized up to 75 degrees C in the presence of K+ ions. The RNA structure is more stable than the equivalent DNA structure (melting temperature approx. 62 degrees C), and its stability is further enhanced in the presence of K+ ions. Two of the central guanine residues are fully protected from cleavage as determined by dimethyl sulphate protection experiments, whereas methylation interference studies show that methylation of any of the four central guanine residues inhibits structure formation. Our results demonstrate that the structured form of the nucleic acid is mediated through the formation of a guanine-tetraplex core region, in RNA this might be further stabilized by the presence of weaker uracil quartets. (+info)
Multiple cloning sites from mammalian expression vectors interfere with gene promoter studies in vitro.
(35/1876)
When performing transcriptional analyses, reporter gene-expression vectors are used to insert promoter fragments through the selected use of a multiple cloning site (MCS) located upstream of the reporter gene. The MCS from pBluescript has frequently been transferred into reporter plasmids (usually bearing the chloramphenical acetyltransferase reporter gene) and used to subclone various promoter fragments from diverse genes. Analyses in electrophoretic mobility shift assay using this MCS as labeled probe revealed that it specifically binds multiple nuclear proteins from a whole array of widely used cell types. Moreover, the presence of the MCS sequence dramatically altered promoter activity in a totally unpredictable fashion that depends on the distance between the MCS and the basal promoter start site of the gene, leading to severe misinterpretation of the transfection data. Finally, we provide evidence that the BamHI/SmaI/PstI restriction site combination is likely one of the major binding site for nuclear proteins on the pBluescript MCS, therefore suggesting that this particular combination of restriction sites should be avoided in the MCS from plasmids that are to be used in promoter studies. (+info)
In vitro transcriptional studies of the bkd operon of Pseudomonas putida: L-branched-chain amino acids and D-leucine are the inducers.
(36/1876)
BkdR is the transcriptional activator of the bkd operon, which encodes the four proteins of the branched-chain keto acid dehydrogenase multienzyme complex of Pseudomonas putida. In this study, hydroxyl radical footprinting revealed that BkdR bound to only one face of DNA over the same region identified in DNase I protection assays. Deletions of even a few bases in the 5' region of the BkdR-binding site greatly reduced transcription, confirming that the entire protected region is necessary for transcription. In vitro transcription of the bkd operon was obtained by using a vector containing the bkdR-bkdA1 intergenic region plus the putative rho-independent terminator of the bkd operon. Substrate DNA, BkdR, and any of the L-branched-chain amino acids or D-leucine was required for transcription. Branched-chain keto acids, D-valine, and D-isoleucine did not promote transcription. Therefore, the L-branched-chain amino acids and D-leucine are the inducers of the bkd operon. The concentration of L-valine required for half-maximal transcription was 2.8 mM, which is similar to that needed to cause half-maximal proteolysis due to a conformational change in BkdR. A model for transcriptional activation of the bkd operon by BkdR during enzyme induction which incorporates these results is presented. (+info)
Directional binding of HMG-I(Y) on four-way junction DNA and the molecular basis for competitive binding with HMG-1 and histone H1.
(37/1876)
Histone H1, HMG-1 and HMG-I(Y) are mammalian nuclear proteins possessing distinctive DNA-binding domain structures that share the common property of preferentially binding to four-way junction (4H) DNA, an in vitro mimic of the in vivo genetic recombination intermediate known as the Holliday junction. Nevertheless, these three proteins bind to 4H DNA in vitro with very different affinities and in a mutually exclusive manner. To investigate the molecular basis for these distinctive binding characteristics, we employed base pair resolution hydroxyl radical footprinting to determine the precise sites of nucleotide interactions of both HMG-1 and histone H1 on 4H DNA and compared these contacts with those previously described for HMG-I(Y) on the same substrate. Each of these proteins had a unique binding pattern on 4H DNA and yet shared certain common nucleotide contacts on the arms of the 4H DNA molecule near the branch point. Both the HMG-I(Y) and HMG-1 proteins made specific contacts across the 4H DNA branch point, as well as interacting at discrete sites on the arms, whereas the globular domain of histone H1 bound exclusively to the arms of the 4H DNA substrate without contacting nucleotides at the crossover region. Experiments employing the chemical cleavage reagent 1, 10-orthophenanthroline copper(II) attached to the C-terminal end of a site-specifically mutagenized HMG-I(Y) protein molecule demonstrated that this protein binds to 4H DNA in a distinctly polar, direction-specific manner. Together these results provide an attractive molecular explanation for the observed mutually exclusive 4H DNA-binding characteristics of these proteins and also allow for critical assessment of proposed models for their interaction with 4H DNA substrates. The results also have important implications concerning the possible in vivo roles of HMG-I(Y), histone H1 and HMG-1 in biological processes such as genetic recombination and retroviral integration. (+info)
Protein footprinting at cysteines: probing ATP-modulated contacts in cysteine-substitution mutants of yeast DNA topoisomerase II.
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Cysteine-substitution mutants of yeast DNA topoisomerase II were used to test footprinting of the enzyme by 2-nitro-5-thiocyanobenzoate, which cyanylates exposed cysteines in a native protein for peptide cleavage at the cyanylated sites upon unfolding and incubating the protein at pH 9. For a mutant enzyme containing a single cysteine, the extent of peptide cleavage was found to reflect the accessibility of the residue in the native protein. For proteins with multiple cysteines, however, such a correlation was obscured by the transfer of cyano groups from modified to unmodified cysteines during incubation of the unfolded protein at pH 9; accessibilities of the cysteinyl residues in a native protein could be assessed only if cyano shuffling was prevented by blocking uncyanylated sulfhydryls with a second thiol reagent. The successive use of two reagents in cysteine footprinting was applied in probing the ATP-modulated formation of contacts in yeast DNA topoisomerase II. (+info)
CIITA-induced occupation of MHC class II promoters is independent of the cooperative stabilization of the promoter-bound multi-protein complexes.
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Precise regulation of MHC class II expression plays a crucial role in the control of the immune response. The transactivator CIITA behaves as a master controller of constitutive and inducible MHC class II gene activation, but its exact mechanism of action is not known. Activation of MHC class II promoters requires binding of at least three distinct multi-protein complexes (RFX, X2BP and NF-Y). It is known that the stability of this binding results from cooperative interactions between these proteins. We show here that expression of CIITA in MHC class II- cells triggers occupation of the promoters by these complexes. This observation raised the possibility that the effect of CIITA on promoter occupation is mediated by an effect on the cooperative stabilization of the DNA-bound multi-protein complexes. We show, however, that the presence of CIITA does not affect the stability of the higher-order protein complex formed on DNA by RFX, X2BP and NF-Y. This suggests other mechanisms for CIITA-induced promoter occupancy, such as an effect on chromatin structure leading to increased accessibility of MHC class II promoters. This ability of CIITA to facilitate promoter occupation is undissociable from its transactivation potential. Finally, we conclude that this effect of CIITA is cell-type specific, since expression of CIITA is not required for normal occupation of MHC class II promoters in B lymphocytes. (+info)
Central role of transcription factor NF-IL6 for cytokine and iron-mediated regulation of murine inducible nitric oxide synthase expression.
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We have previously shown that iron regulates the transcription of inducible nitric oxide synthase (iNOS). To elucidate the underlying mechanisms we performed a series of transient transfections of murine fibroblast (NIH-3T3) and macrophage-like cells (J774.A1) with reporter plasmids containing the iNOS promoter and deletions thereof. By means of this and subsequent DNase I footprinting analysis we identified a regulatory region between -153 and -142 bp upstream of the transcriptional start site of the iNOS promoter that was sensitive to regulation by iron perturbation. Gel shift and supershift assays revealed that the responsible protein for this observation is NF-IL6, a member of the CCAAT/enhancer binding protein family of transcription factors. Binding of NF-IL6 to its consensus motif within the iNOS promoter was inducible by IFN-gamma and/or LPS, was reduced by iron, and was enhanced by the iron chelator desferrioxamine. Introduction of a double mutation into the NF-IL6 binding site (-153/-142) of an iNOS promoter construct resulted in a reduction of IFN-gamma/LPS inducibility by >90% and also impaired iron mediated regulation of the iNOS promoter. Our results provide evidence that this NF-IL6 binding site is of central importance for maintaining a high transcriptional rate of the iNOS gene after IFN-gamma/LPS stimulation, and that NF-IL6 may cooperate with hypoxia inducible factor-1 in the orchestration of iron-mediated regulation of iNOS. (+info)