Relationship among location of T-antigen-induced DNA distortion, auxiliary sequences, and DNA replication efficiency.
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T-antigen-induced DNA distortion was studied in a series of simian virus 40 (SV40) plasmid constructs whose relative replication efficiency ranges from 0.2 to 36. Bending was detected in the wild-type SV40 regulatory region consisting of three copies of the GC-rich 21-bp repeat but not in constructs with only one or two copies of the 21-bp repeat. In a construct with enhanced replication efficiency, bending occurred in a 69-bp cellular sequence located upstream of a single copy of the 21-bp repeat. Bending occurred both upstream of ori and in the three 21-bp repeats located downstream of ori in a construct with reduced replication efficiency. In a construct with no 21-bp repeats, DNA distortion occurred downstream of ori. The results indicate that SV40 DNA replication is enhanced when the structure of the regulatory region allows the DNA to form a bent structure upstream of the initial movement of the replication fork. (+info)
EBNA1 distorts oriP, the Epstein-Barr virus latent replication origin.
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The Epstein-Barr virus nuclear antigen 1 (EBNA1) protein binds and activates the latent replication origin (oriP) of the Epstein-Barr virus. We have been studying EBNA1 to determine how it activates replication at oriP. Here we demonstrate that upon binding of EBNA1 to oriP, two thymine residues become reactive to potassium permanganate (KMnO4), indicating a helical distortion at these sites. The KMnO4-reactive thymines are 64 bp apart in the region of dyad symmetry of oriP. Dimethyl sulfate protection studies indicated that EBNA1 binds on the opposite face of the helix from the reactive thymines. The nature of the helical distortion induced by EBNA1 and its possible significance to the initiation of replication are discussed. (+info)
Spacing is crucial for coordination of domain functions within the simian virus 40 core origin of replication.
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The simian virus 40 core origin of replication is composed of distinct domains that are bracketed by DNA spacers. We created a matched set of insertion mutations in spacer sites to study the spatial relationships among origin domains. Insertions larger than a single base pair severely inhibit replication regardless of the helical phasing between domains. Replication-defective mutations reduce T-antigen binding and T-antigen-induced KMnO4 modifications of DNA to various extents. Mutations in the early half of the origin reduce T-antigen functions in the entire origin, whereas mutations in the late half reduce functions only in that half. Surprisingly, some mutations that severely inhibit DNA replication reduce T-antigen-induced melting and other structural changes within origin DNA to only a limited extent. In contrast, all replication-defective origin mutations prevent T antigen from extending the primary replication bubble beyond the limits of the core origin of replication. We conclude, therefore, that T-antigen-induced events within the core origin must be spatially coordinated for conversion of T-antigen hexamers bound to the core origin into mobile helicase units. (+info)
Interaction of the Bacillus subtilis glnRA repressor with operator and promoter sequences in vivo.
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In vivo dimethyl sulfate footprinting of the Bacillus subtilis glnRA regulatory region under repressing and derepressing conditions demonstrated that the GlnR protein, encoded by glnR, interacts with two sites situated within and adjacent to the glnRA promoter. One site, glnRAo1, between positions -40 and -60 relative to the start point of transcription, is a 21-bp symmetrical element that has been identified as essential for glnRA regulation (H. J. Schreier, C. A. Rostkowski, J. F. Nomellini, and K. D. Hirschi, J. Mol. Biol. 220:241-253, 1991). The second site, glnRAo2, is a quasisymmetrical element having partial homology to glnRAo1 and is located within the promoter between positions -17 and -37. The symmetry and extent of modifications observed for each site during repression and derepression indicated that GlnR interacts with the glnRA regulatory region by binding to both sites in approximately the same manner. Experiments using potassium permanganate to probe open complex formation by RNA polymerase demonstrated that transcriptional initiation is inhibited by GlnR. Furthermore, distortion of the DNA helix within glnRAo2 occurred upon GlnR binding. While glutamine synthetase, encoded by glnA, has been implicated in controlling glnRA expression, analyses with dimethyl sulfate and potassium permanganate ruled out a role for glutamine synthetase in directly influencing transcription by binding to operator and promoter regions. Our results suggested that inhibition of transcription from the glnRA promoter involves GlnR occupancy at both glnRAo1 and glnRAo2. In addition, modification of bases within the glnRAo2 operator indicated that control of glnRA expression under nitrogen-limiting (derepressing) conditions included the involvement of a factor(s) other than GlnR. (+info)
Antp-type homeodomains have distinct DNA binding specificities that correlate with their different regulatory functions in embryos.
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Much of the functional specificity of Drosophila homeotic selector proteins, in their ability to regulate specific genes and to assign specific segmental identities, appears to map within their different, but closely related homeodomains. For example, the Drosophila Dfd and human HOX4B (Hox 4.2) proteins, which have extensive structural similarity only in their respective homeodomains, both specifically activate the Dfd promoter. In contrast, a chimeric Dfd protein containing the Ubx homeodomain (Dfd/Ubx) specifically activates the Antp P1 promoter, which is normally targeted by Ubx. Using a variety of DNA binding assays, we find significant differences in DNA binding preferences between the Dfd, Dfd/Ubx and Ubx proteins when Dfd and Antp upstream regulatory sequences are used as binding substrates. No significant differences in DNA binding specificity were detected between the human HOX4B (Hox 4.2) and Drosophila Dfd proteins. All of these full-length proteins bound as monomers to high affinity DNA binding sites, and interference assays indicate that they interact with DNA in a way that is very similar to homeodomain polypeptides. These experiments indicate that the ninth amino acid of the recognition helix of the homeodomain, which is glutamine in all four of these Antp-type homeodomain proteins, is not sufficient to determine their DNA binding specificities. The good correlation between the in vitro DNA binding preferences of these four Antp-type homeodomain proteins and their ability to specifically regulate a Dfd enhancer element in the embryo, suggests that the modest binding differences that distinguish them make an important contribution to their unique regulatory specificities. (+info)
Mapping of RNA polymerase on mammalian genes in cells and nuclei.
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The assembly of an RNA polymerase II initiation complex at a promoter is associated with the melting of the DNA template to allow the polymerase to read the DNA sequence and synthesize the corresponding RNA. Using the specific single-stranded modifying reagent KMnO4 and a new genomic sequencing technique, we have explored the melted regions of specific genes in genomic DNA of whole cells or of isolated nuclei. We have demonstrated for the first time in vivo the melting in the promoter proximal transcribed region that is associated with the presence of RNA polymerase II complexes. An interferon-inducible gene, ISG-54, exhibited KMnO4 sensitivity over approximately 300 nucleotides downstream of the RNA initiation site in interferon-treated cells when the gene was actively transcribed but not in untreated cells where the gene was not transcribed. The extent of KMnO4 modification was proportional to transcription levels. The KMnO4 sensitivity was retained when nuclei were isolated from induced cells but was lost if the engaged polymerases were further allowed to elongate the nascent RNA chains ("run-on"). The sensitivity to KMnO4 in isolated nuclei was retained if the run-on incubation was performed in the presence of alpha-amanitin, which blocks progress of engaged polymerases. A similar analysis identified an open sequence of only approximately 30 bases just downstream of the start site of the transthyretin (TTR) gene in nuclei isolated from mouse liver, a tissue where TTR is actively transcribed. This abrupt boundary of KMnO4 sensitivity, which was removed completely by allowing engaged polymerases to elongate RNA chains, suggests that most polymerases transcribing this gene paused at about position +20. The possibility of mapping at the nucleotide level the position of actively transcribing RNA polymerases in whole cells or isolated nuclei opens new prospects in the study of transcription initiation and elongation. (+info)
Melting during steady-state transcription of the rrnB P1 promoter in vivo and in vitro.
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The rRNA rrnB P1 promoter was probed with the single-strand-selective reagent potassium permanganate during steady-state transcription in vitro and in vivo. In both cases, a weak but significant level of permanganate sensitivity was observed, which was not changed by treatment with rifampin. In contrast, static studies showed that rifampin strongly affects the very high level signal associated with polymerases that have used ATP and CTP as initiating nucleotides. We infer that the permanganate sensitivity associated with steady-state transcription is due to polymerases that have not yet used ATP and CTP. The slow and regulated step during rrnB P1 transcription may be the use of the initiating nucleotides to catalyze stable opening of the promoter DNA. (+info)
DNA melting within stable closed complexes at the Escherichia coli rrnB P1 promoter.
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Several transcription complexes are shown to form on the E. coli ribosomal rrnB P1 promoter in vitro. These include two closed complexes that are sensitive to heparin attack, and one open complex. The closed complexes are unusual in that they are both highly specific and stable, properties associated with the atypical DNA sequence of this promoter. The effector ppGpp does not prevent closed complex formation but does reduce the level of open complexes that form. (+info)