potassium-permanganate and dimethyl-sulfate

Topics: Alkylating Agents; Base Sequence; DNA; DNA Probes; Furocoumarins; Gene Expression Regulation; Molecular Sequence Data; Osmium Tetroxide; Potassium Permanganate; Protein Binding; Sulfuric Acid Esters; Ultraviolet Rays

Guanine-rich DNAs can fold into four-stranded structures that contain stacks of G-quartets. Bioinformatics studies have revealed that G-rich sequences with the potential to adopt these structures are unevenly distributed throughout genomes, and are especially found in gene promoter regions. With the exception of the single-stranded telomeric DNA, all genomic G-rich sequences will always be present along with their C-rich complements, and quadruplex formation will be in competition with the corresponding Watson-Crick duplex. Quadruplex formation must therefore first require local dissociation (melting) of the duplex strands. Since negative supercoiling is known to facilitate the formation of alternative DNA structures, we have investigated G-quadruplex formation within negatively supercoiled DNA plasmids. Plasmids containing multiple copies of (G3T)n and (G3T4)n repeats, were probed with dimethylsulphate, potassium permanganate and S1 nuclease. While dimethylsulphate footprinting revealed some evidence for G-quadruplex formation in (G3T)n sequences, this was not affected by supercoiling, and permanganate failed to detect exposed thymines in the loop regions. (G3T4)n sequences were not protected from DMS and showed no reaction with permanganate. Similarly, both S1 nuclease and 2D gel electrophoresis of DNA topoisomers did not detect any supercoil-dependent structural transitions. These results suggest that negative supercoiling alone is not sufficient to drive G-quadruplex formation.

Topics: Circular Dichroism; DNA, Superhelical; Fungal Proteins; G-Quadruplexes; Inverted Repeat Sequences; Potassium Permanganate; Single-Strand Specific DNA and RNA Endonucleases; Sulfuric Acid Esters

Potassium permanganate (KMnO4) has widely been used in genomic footprinting assays to map unusual gene structures, including the melting DNA block in transcriptional elongation that results from promoter-proximal pausing of RNA polymerase (Pol) II complexes. Although it has been assumed that DNA-bound proteins do not protect underlying nucleic acids from KMnO4 modifications, we provide evidence herein that this chemical can readily be used to detect nuclear factor loading at a promoter when using optimized conditions. Moreover, by comparing parallel KMnO4 and dimethylsulfate (DMS) in vivo footprintings, we show that the utilization of KMnO4 in combination with another chemical probe maximizes the detection of factor occupancy at a DNA regulatory region, thus providing a better opportunity to define the actual profiles of DNA-protein contacts at given genomic sites in living cells.

Topics: Animals; Base Sequence; DNA; DNA Footprinting; Mice; Molecular Sequence Data; Potassium Permanganate; Promoter Regions, Genetic; Protein Binding; Proteins; Sulfuric Acid Esters; Thymus Gland

We previously reported that gelsolin gene expression is reduced in various tumors. In an effort to gain further insights into the mechanism of gelsolin downregulation in tumors, we examined the in vivo properties of the gelsolin promoter in urinary bladder cancer cell lines. Neither mutation nor hypermethylation was responsible for gene silencing at the promoter. After exposure to trichostatin A (TSA), a histone deacetylase inhibitor, gelsolin promoter activity was markedly enhanced in the cancer cells, not in cells derived from normal tissue. Chromatin immunoprecipitation assays revealed that both histones H3 and H4 were hypoacetylated in the promoter region of the cancer cells, and the accumulation of acetylated histones was detected by TSA treatment. In vivo footprinting analysis revealed the presence of dimethylsulfate (DMS) hypersensitive site in the untranslated region around nucleotide--35 only in the cancer cells but not in cells derived from normal tissue, and analysis of KMnO4 reactive nucleotides showed that the stem loop structure could be formed in vivo of the cancer cells. This novel stem loop structure may play a part in regulating the transcription of the gelsolin gene in the cancer cells. These results suggest that nucleosome accessibility through histone deacetylation and structural changes (DMS hypersensitivity and stem loop structure) in the promoter region form the basis of the mechanism leading to the silencing of gelsolin gene in human bladder cancer.

Topics: Base Sequence; Chromatin; DNA Footprinting; DNA Methylation; Down-Regulation; Gelsolin; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Molecular Sequence Data; Mutagens; Polymerase Chain Reaction; Potassium Permanganate; Precipitin Tests; Promoter Regions, Genetic; Sulfuric Acid Esters; Transcription, Genetic; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Topics: DNA; Electrophoretic Mobility Shift Assay; Molecular Biology; Molecular Probes; Peptide Nucleic Acids; Potassium Permanganate; Sulfuric Acid Esters

We determined the adduct maps of S(N)1 and S(N)2 alkylating agents in cultured human cells (in vivo) and in vitro to probe DNA-protein interactions along sequences of the promoter and exon 1 of the Fragile-X mental retardation 1 (FMR1) gene. Using ligation-mediated polymerase chain reaction (LMPCR), we compared the piperidine-sensitive alkylpurines sites generated by treating cultured cells (in vivo) and naked DNA (in vitro) with S(N)1 (N-methyl-N-nitrosourea, N-nitroso(acetoxymethyl)methylamine and 1-methyl-3-nitro-1-nitrosoguanidine) and S(N)2 alkylating agents (dimethyl sulfate (DMS), methane sulfonic acid methyl ester, iodo methane, diethyl sulfate, methane sulfonic acid ethyl ester and iodo ethane). The FMR1 promoter has four sites where DNA-protein interactions are observed. In these regions, the S(N)1 methylating agent reactions produced only hypo-reactive sites. In contrast, iodoalkane S(N)2 alkylating agents (MeI and EtI) reactions generated only hyper-reactive sites. Although there are hyper-reactive sites for the other S(N)2 reagents, the hyper-reactive site at +14 on the FMR1 map is more pronounced for the sulfate and sulfonate-derived alkylating agents than for the iodoalkanes. However, DMS modification in the presence of methyl sulfone, a compound that does not alkylate DNA, eliminates the hyper-reactive site observed at +14. This suggests that the electron-rich oxygen atoms of the sulfate and sulfonate-derived S(N)2 alkylating agent structure position the alkylating moiety to the neighboring N-7-guanine position to favor alkyl transfer to the guanine. Using KMnO(4) to probe for single-strand DNA, an unpaired cytosine base was detected at the 5'-side of the hyper- reactive guanine base at position +14, consistent with the formation of a local DNA single-strand bulge. In conclusion, we show that the sequence context-dependent formation of alkylpurines is determined by the chemical nature of the alkylating agent, the DNA sequence context, chromatin structure, and the presence of other non-reactive molecules that can inhibit alkylation.

Topics: Alkylating Agents; Alkylation; Base Sequence; Cell Line, Transformed; Chromatin; Dimethyl Sulfoxide; DNA; DNA Damage; DNA Footprinting; DNA Methylation; Exons; Fragile X Mental Retardation Protein; Guanine; Humans; Lymphocytes; Molecular Conformation; Molecular Sequence Data; Nerve Tissue Proteins; Piperidines; Potassium Permanganate; Promoter Regions, Genetic; Purines; RNA-Binding Proteins; Sulfones; Sulfuric Acid Esters

Quantitative analysis of multiple-hit potassium permanganate (KMnO(4)) footprinting has been carried out in vivo on Saccharomyces cerevisiae 5S rRNA genes. The results fix the number of open complexes at steady state in exponentially growing cells at between 8 and 17% of the 150 to 200 chromosomal copies. UV and dimethyl sulfate footprinting set the transcription factor TFIIIB occupancy at 23 to 47%. The comparison between the two values suggests that RNA polymerase III binding or promoter opening is the rate-limiting step in 5S rRNA transcription in vivo. Inhibition of RNA elongation in vivo by cordycepin confirms this result. An experimental system that is capable of providing information on the mechanistic steps involved in regulatory events in S. cerevisiae cells has been established.

Topics: Base Sequence; Chromosomes, Fungal; Deoxyadenine Nucleotides; DNA Footprinting; DNA, Fungal; Molecular Sequence Data; Potassium Permanganate; Promoter Regions, Genetic; RNA Polymerase III; RNA, Fungal; RNA, Ribosomal, 5S; Saccharomyces cerevisiae; Sulfuric Acid Esters; Transcription Factor TFIIIB; Transcription Factors; Transcription, Genetic; Ultraviolet Rays

We have investigated interaction of Mycobacterium smegmatis topoisomerase I at its specific recognition sequence. DNase I footprinting demonstrates a large region of protection on both the scissile and non-scissile strands of DNA. Methylation protection and interference analyses reveal base-specific contacts within the recognition sequence. Missing contact analyses reveal additional interactions with the residues in both single and double-stranded DNA, and hence underline the role for the functional groups associated with those bases. These interactions are supplemented by phosphate contacts in the scissile strand. Conformation specific probes reveal protein-induced structural distortion of the DNA helix at the T-A-T-A sequence 11 bp upstream to the recognition sequence. Based on these footprinting analyses that define parameters of topoisomerase I-DNA interactions, a model of topoisomerase I binding to its substrate is presented. Within the large protected region of 30 bp, the enzyme makes direct contact at two locations in the scissile strand, one around the cleavage site and the other 8-12 bases upstream. Thus the enzyme makes asymmetric recognition of DNA and could carry out DNA relaxation by either of the two proposed mechanisms: enzyme bridged and restricted rotation.

Topics: Base Sequence; Binding Sites; Deoxyribonuclease I; DNA; DNA Footprinting; DNA Methylation; DNA Topoisomerases, Type I; DNA-Binding Proteins; Models, Molecular; Mycobacterium; Nucleic Acid Conformation; Phosphates; Potassium Permanganate; Protein Binding; Rotation; Static Electricity; Substrate Specificity; Sulfuric Acid Esters

Topics: CDC2 Protein Kinase; Cells, Cultured; Chromatin; Deoxyribonuclease I; DNA; DNA Footprinting; DNA Methylation; DNA Nucleotidylexotransferase; DNA Primers; Electrophoresis, Agar Gel; Electrophoresis, Polyacrylamide Gel; Fibroblasts; HeLa Cells; Humans; Indicators and Reagents; Polymerase Chain Reaction; Potassium Permanganate; Sulfuric Acid Esters

Topics: Animals; Cell Membrane Permeability; Cell Nucleus; Chromatin; Cross-Linking Reagents; Deoxyribonuclease I; DNA; Indicators and Reagents; Nucleosomes; Polymerase Chain Reaction; Potassium Permanganate; Restriction Mapping; Sulfuric Acid Esters; Ultraviolet Rays

The Bradyrhizobium japonicum fixRnifA operon is transcribed from two promoters: fixRp1, a -24/-12 promoter recognized by the sigma54-holoenzyme form of the RNA polymerase, and fixRp2, a -35/-10 promoter that is transcribed by a second, unidentified, form of RNA polymerase holoenzyme. The fixRp1 promoter is autoregulated during microaerobiosis by NifA, whereas fixRp2 is also activated, but by a different regulatory protein. The main transcription start sites for these promoters are just two nucleotides apart, such that the conserved -12 and -10 regions of fixRp1 and fixRp2, respectively, must overlap each other, whereas the -24 and -35 regions lie one DNA helical turn apart. Using in vivo genomic dimethyl sulfate and KMnO4 footprinting, we showed that the promoter region is differentially protected, depending upon which holoenzyme is bound. Mutagenesis analyses indicated that positions from -12 to -14 are critical for the activity of both promoters, whereas mutations at -10 and -11 affected mainly fixRp2 expression. When the sequence of the putative -35 region of fixRp2 was modified to match the putative consensus, expression from this promoter was increased 3-fold and the reactivity toward KMnO4, but not the transcriptional start site, moved two nucleotides further upstream, indicating that the altered promoter forms a different open complex. Additionally, we detected NifA-dependent methylation protection of two atypical NifA binding sites and protection of guanine -75. The latter residue is located in a region critical for fixRp2 promoter activation. The results present direct physical evidence of the complexity of the organization, regulation, and function of the fixRnifA promoter region.

Topics: Bacterial Proteins; Base Sequence; Chromosome Mapping; DNA Footprinting; DNA Methylation; DNA-Directed RNA Polymerases; DNA, Bacterial; Isomerism; Molecular Sequence Data; Mutagenesis; Oxidoreductases; Potassium Permanganate; Promoter Regions, Genetic; Rhizobiaceae; Sulfuric Acid Esters; Transcription Factors

An in organello footprinting approach has been used to probe a protein-DNA interaction of a nuclear coded 25 kDa protein, previously isolated in our laboratory, that binds "in vitro" a region within the ND2 gene, located upstream of the Ori-L. Footprinting studies with the purine-modifying reagent dimethyl sulfate and the pirimidine-modifying reagent potassium permanganate were carried out in isolated mitochondria from rat liver. Dimethyl sulfate footprinting has allowed the detection of a protein-DNA interaction within the curved ND2 region with contact sites located in both the strands. Potassium permanganate footprinting allowed detection of an adjacent permanganate-reactive region. We hypothesize that the permanganate-reactive region is a single stranded DNA due to a profound helix distortion induced by a 25 kDa protein binding to the nearest region.

Topics: Animals; Base Sequence; Deoxyribonucleoproteins; DNA Footprinting; DNA Replication; DNA-Binding Proteins; DNA, Mitochondrial; Mitochondria, Liver; Molecular Weight; Nucleic Acid Conformation; Potassium Permanganate; Protein Binding; Rats; Sulfuric Acid Esters

The Escherichia coli RecBCD enzyme unwinds DNA from a free double-stranded DNA end to produce single-stranded DNA intermediates of homologous recombination. In the absence of ATP RecBCD binds to a free DNA end to form an initiation complex for DNA unwinding. We studied the structure of these complexes formed with blunt-ended, 5'-extended, and 3'-extended DNA. Reactivity to the single-stranded DNA-specific reagents KMnO4 and dimethyl sulfate indicated that RecBCD opened, in a Mg(2+)-dependent manner, the terminal five or six base-pairs in each substrate. Thymine residues located four to six nucleotides from the 5' end were only partially reactive to KMnO4, suggesting that part of the 5'-terminated strand was partially shielded by the enzyme. DNase I footprinting indicated that the enzyme positions itself relative to the end of the longer of the two strands, although an exception was noted. These results imply flexibility in the ability of RecBCD to open the DNA and position itself for unwinding on DNA with different types of ends. They also imply conformational differences of RecBCD enzyme bound to different types of ends; these conformational differences may be related to those occurring during the unwinding cycle.

Topics: Adenosine Triphosphate; Deoxyribonuclease I; DNA; DNA Footprinting; DNA Helicases; DNA, Single-Stranded; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Exodeoxyribonuclease V; Exodeoxyribonucleases; Hydrazines; Hydroxyl Radical; Magnesium; Models, Chemical; Nucleic Acid Conformation; Potassium Permanganate; Sulfuric Acid Esters

Topics: Animals; Cell Fractionation; Cell Nucleus; Cells, Cultured; Deoxyribonuclease I; DNA; DNA-Binding Proteins; Genome; HeLa Cells; Humans; Indicators and Reagents; Leukocytes; Mutagenesis; Mutagens; Polymerase Chain Reaction; Potassium Permanganate; RNA Probes; Sulfuric Acid Esters; Templates, Genetic

The binding of PNA (peptide nucleic acid) T2CT2CT4-LysNH2 to the double-stranded DNA target 5'-A2GA2GA4 was studied by KMnO4 and dimethylsulfate (DMS) probing. It is found that upon sequence-specific strand displacement binding of the PNA to the dsDNA target concomitant protection of the N-7 of guanines within the target takes place. It is furthermore shown that the binding of this PNA is more efficient at pH 5.5 than at pH 6.5 and very inefficient at pH 7.5. These results clearly indicate that C+G Hoogsteen base pairing is present and important for binding and that the strand displacement complex therefore involves a PNA.DNA-PNA triplex.

Topics: Base Sequence; Cytosine; DNA; Guanine; Hydrogen Bonding; Hydrogen-Ion Concentration; Molecular Sequence Data; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Oligonucleotide Probes; Plasmids; Potassium Permanganate; Pyrimidine Nucleotides; Sulfuric Acid Esters

Telomeric DNA of a variety of vertebrates including humans contains the tandem repeat d(TTAGGG)n. We have investigated the structural properties of the human telomeric repeat oligonucleotide models d(T2AG3)4, d(G3T2A)3G3, and d(G3T2AG3) using CD, gel electrophoresis, and chemical probing techniques. The sequences d(G3T2A)3G3 and d(T2AG3)4 assume an antiparallel G quartet structure by intramolecular folding, while the sequence d(G3T2AG3) also adopts an antiparallel G quartet structure but by dimerization of hairpins. In all the above cases, adenines are in the loop. The TTA loops are oriented at the same end of the G tetrad stem in the case of hairpin dimer. Further, the oligonucleotide d(G3T2AG3) forms a higher order structure by the association of two hairpin dimers via stacking of G tetrad planes. Here we show that N-7 of adenine in the hairpin dimer is Hoogsteen hydrogen-bonded. The partial reactivity of loop adenines with DEPC in d(T2AG3)4 suggests that the intramolecular G quartet structure is highly polymorphic and structures with different loop orientations and topologies are formed in solution. Intra- and interloop hydrogen bonding schemes for the TTA loops are proposed to account for the observed diethyl pyrocarbonate reactivities of adenines. Sodium-induced G quartet structures differ from their potassium-induced counterparts not only in stability but also in loop conformation and interactions. Thus, the overall structure and stability of telomeric sequences are modulated by the cation present, loop sequence, and the number of G tracts, which might be important for the telomere function.

Topics: Circular Dichroism; Diethyl Pyrocarbonate; Humans; Hydrogen Bonding; Models, Molecular; Nucleic Acid Conformation; Nucleic Acid Denaturation; Oligonucleotides; Potassium; Potassium Permanganate; Repetitive Sequences, Nucleic Acid; Sodium; Sulfuric Acid Esters; Telomere; Thermodynamics

RecA protein from Escherichia coli has been used to form a triple-stranded DNA structure from either single-stranded M13 DNA or a single-stranded oligonucleotide plus a duplex oligonucleotide with a hairpin loop. The secondary structure of purified deproteinized triplex was examined by probing with DNase I, P1 nuclease, potassium permanganate, and diethyl pyrocarbonate. The two strands destined to form heteroduplex DNA showed the same patterns of chemical modification and enzymatic digestion as control duplex DNA, indicating that they formed a normal duplex substructure. However, the nascent outgoing strand showed properties consistent with a novel triplex structure: most of its purine residues, especially adenines, were hyperreactive to all probes. The patterns of digestion by DNase I and P1 nuclease indicated that the nascent outgoing strand was not a freely mobile or single-stranded branch but rather was still interacting with the newly formed heteroduplex DNA. On the basis of the planar base triads proposed previously (Rao et al., 1993) and energy minimization of a third strand in the major groove of B-form DNA, we derived a model that helps to rationalize the properties revealed by chemical and enzymatic probing.

Topics: Bacteriophage M13; Base Sequence; Diethyl Pyrocarbonate; DNA; DNA, Viral; Hydrogen Bonding; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Potassium Permanganate; Rec A Recombinases; Sulfuric Acid Esters

The mechanism by which DNA helicases unwind DNA was tested; an "unwinding complex" between the SV40 large tumor antigen (T antigen) and a DNA molecule designed to resemble a replication fork was probed. In an adenosine triphosphate (ATP)-dependent reaction, T antigen quantitatively recognized this synthetic replication fork and bound the DNA primarily as a hexamer. The T antigen bound only one of the two strands at the fork, an asymmetric interaction consistent with the 3'----5' directionality of the DNA helicase activity of T antigen. Binding to chemically modified DNA substrates indicated that the DNA helicase recognized the DNA primarily through the sugar-phosphate backbone. Ethylation of six top strand phosphates at the junction of single-stranded and double-stranded DNA inhibited the DNA helicase activity of T antigen. Neither a 3' single-stranded end on the DNA substrate nor ATP hydrolysis was required for T antigen to bind the replication fork. These data suggest that T antigen can directly bind the replication fork through recognition of a fork-specific structure.

Topics: Adenosine Triphosphate; Antigens, Polyomavirus Transforming; Diethyl Pyrocarbonate; DNA Helicases; DNA Replication; DNA, Single-Stranded; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Ethylnitrosourea; Formates; Potassium Permanganate; Sulfuric Acid Esters; Time Factors

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.

Topics: Bacillus subtilis; Base Sequence; Binding Sites; DNA-Binding Proteins; Gene Expression Regulation, Bacterial; Glutamate-Ammonia Ligase; Guanine; Methylation; Molecular Sequence Data; Operator Regions, Genetic; Potassium Permanganate; Promoter Regions, Genetic; Pyrimidines; Regulatory Sequences, Nucleic Acid; Repressor Proteins; Sulfuric Acid Esters

The FLP protein of the 2 microns plasmid of Saccharomyces cerevisiae promotes conservative site-specific recombination between DNA sequences that contain the FLP recognition target (FRT). FLP binds to each of the three 13 base pair symmetry elements in the FRT site in a site-specific manner. We have probed both major and minor groove contacts of FLP using dimethyl sulphate, monoacetyl-4-hydroxyaminoquinoline 1-oxide and potassium permanganate and find that the protein displays extensive interactions with residues of both the major and minor grooves of 10 base pairs of each symmetry element. We find no evidence that the FRT site assumes a single-stranded conformation upon FLP binding.

Topics: 4-Hydroxyaminoquinoline-1-oxide; Base Sequence; Binding Sites; DNA Nucleotidyltransferases; DNA, Fungal; DNA, Single-Stranded; Fungal Proteins; Guanine; Methylation; Molecular Sequence Data; Potassium Permanganate; Restriction Mapping; Saccharomyces cerevisiae; Substrate Specificity; Sulfuric Acid Esters; Thymine

Topics: Chromatography, Gel; Chromosomes, Bacterial; DNA; DNA-Binding Proteins; DNA, Bacterial; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Indicators and Reagents; Oligodeoxyribonucleotides; Plasmids; Polymerase Chain Reaction; Potassium Permanganate; Sulfuric Acid Esters

Activation of transcription at the Klebsiella pneumoniae nifLA promoter requires the phosphorylated form of the positive control protein NTRC, together with RNA polymerase modified by the alternative sigma factor sigma 54. Dimethylsulphate and potassium permanganate were used as probes to analyse the interaction of NTRC and sigma 54-RNA polymerase with supercoiled nifLA promoter DNA in vitro. In contrast to the glnAp2 promoter, sigma 54 holoenzyme did not protect guanine residues in the nifLA promoter from methylation in the absence of the activator. We propose that NTRC stabilizes the interaction of sigma 54-RNA polymerase with the -24, -12 region, in addition to its role in catalysing open complex formation. Phosphorylated NTRC binds to two sites located greater than 100 nucleotides upstream of the -24, -12 region; it also induces hyper-methylation of a G residue at -23. Enhanced methylation at -23 is not co-operative with the binding of activator to the upstream sites and may account for the ability of NTRC, when present at high concentration, to activate transcription in the absence of the upstream binding sites. The insertion of spacer mutations at -86 indicates that transcriptional activation of the nifLA promoter at low NTRC concentrations is face-of-the-helix dependent, both in vivo and in vitro. We propose that correct positioning of activator molecules at the upstream binding sites stabilizes the interaction of sigma 54-RNA polymerase with the downstream region via the formation of a DNA loop.

Topics: Bacterial Proteins; Base Sequence; Chromosome Mapping; DNA-Binding Proteins; DNA-Directed RNA Polymerases; Genes, Bacterial; Klebsiella pneumoniae; Molecular Sequence Data; Nitrogen Fixation; Phosphorylation; PII Nitrogen Regulatory Proteins; Plasmids; Potassium Permanganate; Promoter Regions, Genetic; Sigma Factor; Sulfuric Acid Esters; Trans-Activators; Transcription Factors; Transcription, Genetic