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Summary MNase-seq (micrococcal nuclease sequencing) is used to map nucleosome positions in eukaryotic genomes to study the relationship between chromatin structure and DNA-dependent processes. Current protocols require at least two days to isolate nucleosome-protected DNA fragments. We have developed a streamlined protocol for S. cerevisiae and other fungi which takes only three hours. Modified protocols were developed for wild fungi and mammalian cells. This method for rapidly producing sequencing-ready nucleosome footprints from several organisms makes MNase-seq faster and easier, with less chemical waste., Graphical abstract, Highlights • A fast way to prepare micrococcal nuclease nucleosome footprints for MNase-seq • Eliminates use of phenol and chloroform and reduces the amount of cells required • Adaptable for a variety of organisms, MNase-seq is used to map nucleosome positions in eukaryotic genomes to study the relationship between chromatin structure and DNA-dependent processes. Current protocols require at least two days to isolate nucleosome-protected DNA fragments. We have developed a streamlined protocol for S. cerevisiae and other fungi which takes only three hours. Modified protocols were developed for wild fungi and mammalian cells. This method for rapidly producing sequencing-ready nucleosome footprints from several organisms makes MNase-seq faster and easier, with less chemical waste.

The expressions of type I and type II macrophage scavenger receptors (MSRs) are highly specific in macrophages and related cell types. Although some reports have described the regulation of MSR gene expression and proposed some cis-elements related to cell-specific expression, the regulation of MSR remains largely unclear. This is due, in part, to an unacceptably low efficiency of transfection into monocyte/ macrophage cells. In the present study, we optimized the conditions of electroporation in murine macrophage (P388D1) cells. The efficiency of electroporation was increased 20-fold compared with previous methods. Using the optimized method, we focused on studying the regulation of the human MSR promoter in macrophages. We presently demonstrate that: a) the proximal -10 to +50 bp human MSR promoter region is necessary for the cell type-specific expression of human MSR; b) the 6.5 kbp upstream sequence suppresses the expression of human MSR; c) a promoter region extending from -504 to -399 bp produced the greatest increase in transcriptional activity; d) macrophage cell-specific transcription factors bind to the region as determined by electrophoretic mobility shift assay (EMSA) and a footprint assay; and e) mutations of the region reduced about 40-75% of the promoter activity in a transfecting assay. We concluded that novel elements located at the -504 to -399 bp region may play an important role in the regulation of the MSR gene expression in macrophages. We speculate that macrophage-specific factors binding to those elements may be responsible for the transcription regulation of the MSR gene in macrophages.

Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity., Graphical abstract, Highlights • Long TIR is a trait found in different superfamilies of DNA transposons. • Long TIR increases the length of the transposon decreasing transposition efficiency. • We have reconstructed functional protein sequences of the long-TIR Galileo element. • Multiple transposase binding sites have been found in the long Galileo TIR. • Long TIR multiple binding sites may offset the negative effect of transposon length.

International audience; Ss-LrpB is a transcription factor of the archaeon Sulfolobus solfataricus that belongs to the leucine-responsive regulatory protein family. This protein binds to three distinct binding sites in the control region of its own gene, suggestive of autoregulation. Here, we present a detailed study of the thermodynamic and conformational rules that govern the interaction between Ss-LrpB and its tripartite operator DNA. Lane-per-lane partition analysis of macroscopic binding state populations in electrophoretic mobility shift assays, probing binding to full-length, truncated and mutated forms of the operator, allowed determination of equilibrium association constants and cooperativity parameters. The resulting thermodynamic model demonstrates that the Ss-LrpB-operator regulatory complex is formed with a significant positive cooperativity, which is mostly arising from dimer-dimer interactions between pairs of adjacent binding sites. There is a constraint on the spacing between these binding sites, with a preference for a cis-alignment on the DNA helix and with a 16-bp linker yielding maximal pairwise cooperativity. DNase I footprinting assays demonstrated that the extent of Ss-LrpB-induced DNA deformations depends on linker length. The knowledge of the thermodynamic principles underlying the Ss-LrpB-operator interaction, presented here, will contribute to unraveling of the cis-regulatory code of Ss-LrpB autoregulation.

Ru-pac complexes (pac = polyaminecarboxylate) are prospective in many ways for biological applications. They have unique features that make them suitable for biological applications. For instance, Ru-pac complexes can bind to biomolecules through a rapid and facile aqua-ligand substitution reaction and have a range of accessible and stable oxidation states. Ru-pac complexes also have some notable catalytic properties that mimic the enzymatic hydrocarbon oxidation by cytochrome P450 under homogeneous conditions, which is of immense significance toward developing Ru-pac-based agents for oxidative cleavage of DNA and artificial nuclease in DNA footprinting experiments. The advancement of Ru-pac-mediated bioinorganic reactions in terms of unraveling the mechanistic information has not been systematically reviewed till date. Hence, the subject of this review comprises mostly kinetic and mechanistic studies of bioinorganic reactions involving Ru-pac complexes. This review highlights the mechanistic aspects of recent investigations on the reactions of Ru-pac complexes that emphasize the prospect of the biological application of such complexes and ascertain the likely mechanisms of action of the Ru-pac complexes in some bioinorganic processes.

Hydroxyl-radical footprinting utilizes the ability of a highly reactive species to nonspecifically cleave the solvent accessible regions of a nucleic acid backbone. Thus, changes in a nucleic acids structure can be probed either as a function of time or of a reagent's concentration. When combined with techniques that allow single nucleotide resolution of the resulting fragments, footprinting experiments provide richly detailed information about local changes in tertiary structure of a nucleic acid accompanying its folding or ligand binding. In this chapter, we present two protocols of equilibrium hydroxyl-radical footprinting based on peroxidative and oxidative Fenton chemistry and discuss how to adjust the Fenton reagent concentrations for a specific experimental condition. We also discuss the choice of the techniques to separate the reaction products and specifics of the data analysis for equilibrium footprinting experiments. Protocols addressing the use of peroxidative Fenton chemistry for time-resolved studies have been published [Schlatterer and Brenowitz, 2009. Methods; Shcherbakova and Brenowitz, 2008. Nat. Protoc.3(2), 288-302; Shcherbakova et al., 2006. Nucleic Acids Res.34(6), e48; Shcherbakova et al., 2007. Methods Cell Biol.84, 589-615].

The promoter of the murine probasin (PB) gene exhibits strong androgen receptor (AR)-specific and tissue-specific regulation and is considered a promising candidate for gene therapy treatment of advanced prostate cancer. To characterize the determinants of chromatin specificity of the PB promoter with the AR we initially investigated the in vitro interactions of recombinant AR DNA binding domain (AR-DBD) with reconstituted nucleosomes incorporating the proximal PB promoter (nucleotides −268 to −76). We demonstrate that a DNA fragment of this promoter region exhibits strong nucleosome positioning. The phased DNA sequence protected by the histone octamer includes four androgen receptor response elements (AREs) which are arranged as two sets of class I and class II sites spaced approximately 90 bp apart. Class I AREs form classical contacts with the AR, whereas class II AREs contain atypical binding sequences and have been shown to stabilize AR binding to adjacent class I sites, resulting in synergistic transcriptional activation and increased hormone sensitivity. We used DNase 1 footprinting and electrophoretic mobility shift assays (EMSA) to show that the AR-DBD binds to its cognate sequences independently of their nucleosomal organization. In addition, we show that the ability of the AR-DBD to interact with the nucleosomal PB promoter is not affected by histone acetylation. Thus the AR-DBD is able to bind to its cognate sequences within the PB promoter in a way that is indifferent to the presence or absence of histones and nucleosomal structure.

In the last decade, the efficiency of DNA sequencing has increased dramatically, making it possible to sequence the entire human genome and genomes of many other organisms. This advance featured the development of sensitive fluorescence resonance energy transfer (FRET)-based dye terminators, DNA polymerases that incorporate these terminators very efficiently and high-throughput capillary electrophoresis instruments. Further improvements may be obtained using improved FRET dyes and charge-modified nucleotides for “direct-loading” of sequencing reaction products. The chemistry of FRET-based dye-nucleotide terminators, charge-modified nucleotides (negative as well as positive) and terminal phosphate-labeled nucleotides for DNA sequencing is discussed in this review.

TtgR is the specific transcriptional repressor of the TtgABC efflux pump. TtgR and the TtgB efflux pump proteins possess multidrug-binding capacity, and their concerted action is responsible for the multidrug resistance phenotype of Pseudomonas putida DOT-T1E. TtgR binds to a pseudo-palindromic site that overlaps the ttgR/ttgA promoters. Dimethylsulfate footprint assays reveal a close interaction between TtgR and the central region of this operator. The results of analytical ultracentrifugation demonstrate that TtgR forms stable dimers in solution, and that two dimers bind to the operator. Microcalorimetric analysis of the binding of the two TtgR dimers to the cognate operator showed biphasic behavior, and an interaction model was developed for the cooperative binding of two TtgR dimers to their target operators. The binding of the two TtgR dimers to the operator was characterized by a Hill coefficient of 1.63 ± 0.13 (kD = 18.2( ± 6.3) μM, kD′ = 0.91( ± 0.49) μM), indicating positive cooperativity. These data are in close agreement with the results of sedimentation equilibrium studies of TtgR–DNA complexes. A series of oligonucleotides were generated in which the imperfect palindrome of the TtgR operator was empirically optimized. Optimization of the palindrome did not significantly alter the binding of the initial TtgR dimer to the operator, but increased the cooperativity of binding and consequently the overall affinity. The minimal fragment for TtgR binding was a 30-mer DNA duplex, and analysis of its sequence revealed two partially overlapping inverted repeats co-existing within the large pseudo-palindrome operator. Based on the architecture of the operator, the thermodynamics of the process, and the TtgR–operator interactions we propose a model for the binding of TtgR to its target sequence., This study was supported by grants BIO2003-00515 and BIO2006-05668 from the CICYT, and by grants CVI-344 and CVI-1912 from Junta de Andalucía.

Publisher Summary This chapter describes a simple temperature-dependent fluorescence assay of the gradual conformation changes of RNAP in these intermediates. Assays involve a wide range of temperature from 4o to 37oC in order to probe as many intermediate states as possible. Simple fluorescence assays to monitor the conformational changes of RNAP during the formation of an open complex at trp promoter are described. The assays are more informative if RNAP with different σ subunits along with the different promoter mutants with known biochemical phenotypes. Another interesting application studies the effect of repressor and activator molecules on RNAP–promoter combinations. It is expected that the fluorescence properties of either the active site or the whole RNAP alter in response to the repressor and activator molecules. Finally, as fluorescence signals are indirect measurements of conformational changes, it is important to use these techniques on biochemically characterized RNAP–promoter complexes. If these were to be applied to a new system, it is advisable to perform parallel biochemical assays such as DNA footprinting to confirm the existence of intermediate states.

Publisher Summary This chapter observes the principles and methods of affinity cleavage in studying transcription. The development of chemical reagents capable of cleaving protein and/or nucleic acid backbones has provided the means to probe the structures of complex biomolecular systems in solution. One useful technique includes iron-EDTA (ethylenediaminetetraacetate) cleavage, which takes place in the presence of an oxidizing agent such as hydrogen peroxide (H2O2) and a reducing agent such as ascorbate (Vitamin C). The products of redox chemistry at the iron center can induce the cleavage of nearby protein, DNA, and RNA backbones. This cleavage activity of iron-EDTA has proved useful for the identification of protein-binding sites on DNA (DNA footprinting) and, more recently, the binding surfaces of proteins (protein foot printing). Conjugation of the cutting reagent to a site on a biological molecule provides a more powerful way to map proximity relations within or between macromolecules. Preparing a large enough set of single-Cys mutants allow mapping the entire periphery of a protein-protein interaction site. However, an abbreviated procedure that approaches this goal without molecular cloning is discussed

Publisher Summary This chapter describes quantitative footprinting methods to study the binding of the aminoglycoside drug paromomycin to a 176-nt segment of RNA from the packaging region of HIV-1 (LAI). In the footprinting experiment, the binding sites of a drug or ligand on a DNA or RNA fragment are determined by cleaving the fragment, which has previously been allowed to come to equilibrium with the ligand, and measuring the amounts of oligomers of various lengths produced in the cutting reaction. The most convenient way of setting up the footprinting experiment is to radiolabel the original fragment at one end and detect labeled oligomers only, so that from the length of a detected oligomer the position at which cleavage took place can be determined. The footprinting experiment involves (1) establishing equilibrium between the labeled RNA and the ligand, (2) cleaving the RNA, (3) separating the products of cleavage in a sequencing gel, (4) measuring intensities of bands corresponding to different-length fragments, and (5) comparing the intensities obtained in the control experiment, in which no ligand is present, with those obtained in experiments with ligand.

Publisher Summary This chapter discusses high-resolution footprinting techniques of drug–DNA complexes using chemical and enzymatic probes. Footprinting is a method that has been developed for determining the sequence-specific binding of small molecules, oligonucleotides, or proteins to DNA. The technique, which was originally designed for analyzing protein–DNA interactions, is based on the ability of ligands to protect DNA from enzymatic or chemical cleavage at their binding sites. Several enzymatic and chemical agents have been developed as footprinting probes, and each has its own characteristic advantages and disadvantages. Enzymes, such as DNase I, are often easy to use but typically overestimate the size of the footprint (on account of their size) and generate uneven cleavage patterns in the absence of added ligand. Chemical agents, such as dimethyl sulfate, osmium tetroxide, or diethyl pyrocarbonate (DEPC), are of limited usefulness because they react only with specific DNA bases. Other chemical probes such as methidiumpropyl–EDTA–Fe(II) [MPE–Fe(II)] act by intercalation and so perturb the DNA structure. Arguably the best cleavage agent for accurately mapping small molecule binding sites on DNA is the hydroxyl radical.

Publisher Summary This chapter describes fluorescence spectroscopy as an established technique for the analysis of macromolecular structure and dynamics and, in more recent studies, is emerging as a promising method for the study of DNA-protein interactions. The chief advantage of fluorescence-based approaches over the gel electrophoretic and filter-binding methods commonly used to characterize DNA protein interactions is the ability to monitor binding events in solution under conditions of true thermodynamic equilibrium. The chapter reviews the application of time-resolved fluorescence spectroscopy to the study of DNA-protein interactions. It provides an overview of general principles, focusing on those time-resolved fluorescence techniques that are most pertinent to the study of DNA-protein interactions. It discusses the instrumentation required for time-resolved fluorescence measurements and the methods employed in site-specific fluorescent labelling of DNA. A number of representative studies are then presented to demonstrate the different experimental strategies that can be used to characterize DNA-protein complexes. The specific examples presented in the chapter describe the ability of time-resolved fluorescence techniques to yield both structural and energetic information on DNA protein interactions.

The transcription program of the Bacillus phage GA-1, a distant relative of phage Φ29, has been studied. Transcription of the GA-1 genome occurred in two stages, early and late. Early genes were expressed from two promoters equivalent to the Φ29 A2b and A2c promoters, whereas late transcription started at a site equivalent to the Φ29 late A3 promoter. The activity of the GA-1 early A2b and A2c promoters diminished 10 minutes after infection, a time at which expression of the late promoter increased significantly. The switch from early to late transcription required protein synthesis, suggesting the need for viral protein(s). An open reading frame was found in the GA-1 genome coding for a protein showing a 53 % similarity to Φ29 regulatory protein p4, and was named p4G. In Φ29, protein p4 represses the early A2b and A2c promoters and activates the late A3 promoter by recruiting RNA polymerase to it. A binding site for protein p4G was localized upstream from the GA-1 late A3 promoter, overlapping with the early A2b promoter. In vitro, protein p4G prevented the binding of RNA polymerase to the GA-1 early A2b promoter but, unlike in Φ29, had no effect on the expression of the late A3 promoter: RNA polymerase could efficiently bind and initiate transcription from the A3 promoter in the absence of protein p4G. Therefore, activation of late transcription occurs differently in GA-1 and Φ29. We propose that protein p4G is an anti-repressor which inhibits the binding to the late promoter of an unknown repressor factor present in the host strain., This investigation has been aided by Research Grant 5R01 GM27242–19 from the National Institutes of Health, by Grant PB93-0173 from Dirección General de Investigación Cientı́fica y Técnica, and by an Institutional Grant from Fundación Ramón Areces to the Centro de Biologı́a Molecular “Severo Ochoa”. J.A.H. and M.M. were holders of pre-doctoral fellowships from Comunidad Autónoma de Madrid.

Publisher Summary Chromosomal proteins that affect the maintenance, propagation, and expression of the genome often interact with DNA only indirectly through other DNA-binding factors. This chapter describes a method that helps determine the position at which such factors interact directly or indirectly with DNA in the genome. The yeast Saccharomyces cerevisiae is used for this purpose because of its wholly sequenced genome and the ease by which it is possible to introduce targeted mutations in specific genes. The initial step is the cross-linking of live cells. Formaldehyde (FA) is a reagent particularly useful for this purpose; it has long been used in studies of histone organization in the nucleosome or protein–DNA interactions. Thus, cross-linking can be done with intact cells, which reduces the risk of redistribution or reassociation of chromosomal proteins during the preparation of cellular or nuclear extracts. Finally, the polymerase chain reaction (PCR) products are analyzed by polyacrylamide gel electrophoresis. The chapter also discusses immunoprecipitation and DNA isolation.

The analysis of functional DNA regulatory sequences involved in transcriptional control is critical to establishing which proteins mediate cell-specific gene expression. The organization of erythroid LCRs is complex, consisting of multiple, interdigested cis elements. As in situ binding to these sites is determined by the accessibility of these regulatory regions in native chromatin and the availability of relevent cell-specific and ubiquitous factors, in vivo footprinting was used to define protein DNA interactions in human globin LCRs.19,20 To further enhance the detection of protein contacts with this technique, we have modified the dimethyl sulfate-based ligation-mediated PCR in vivo footprinting procedure to permit the assessment of protein binding at guanine and adenine resides, rather than exclusively at guanines. This modification, termed GA-LMPCR in vivo footprinting, was essential for the analysis of GATA-1 motifs in the α-LCR and HS-3 of the β-LCR. Moreover, GA-LMPCR in vivo footprinting provided high-resolution analysis of AP-1/NF-E2 elements and revealed protein contacts at sequences that are not coincident with previously described regulatory motifs. A comprehensive discussion of this modification and sample illustrations from our studies have been presented to demonstrate the enhanced detection and resolution obtained with this procedure.

A new regulatory region (-108 to -86), named CE, containing potential CRE- and Ets-binding sites has been identified in the murine E-cadherin promoter. The Ets-binding site (at -97 position) negatively modulates the activity of the E-cadherin promoter in expressing keratinocyte cell lines and was responsible for the specific retarded complexes obtained with the CE region. Analysis of the methylation status of the endogenous E-cadherin promoter indicated that silencing of E-cadherin expression in malignant keratinocytes cannot be explained by hypermethylation mechanisms. Furthermore, treatment with 5'-aza-2'-deoxycytidine was unable to induce the expression of E-cadherin in deficient keratinocytes. However, in vivo footprinting analysis of the endogenous E-cadherin promoter showed a very distinct pattern in expressing and nonexpressing keratinocytes. Extensive interactions in the previously postulated proximal regulatory elements and in the CE region were detected in expressing cells, while only some nucleotides of the E-pal element and of the CE region were protected in nonexpressing keratinocytes. These results indicate a complex regulation of the mouse E- cadherin promoter and support a model where the combination of positive (CCAAT-box and GC-rich region) and negative (E-pal element and CE region) cis-acting elements contribute to the final level of E-cadherin gene expression. In addition, our results show that down-regulation of E-cadherin expression in transformed epidermal keratinocytes is mainly exerted through the interaction of repressor factor(s) with the E-pal element and to the lack of interaction of positive acting factors with the proximal regions., This work was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (SAF95-0818, SAF98-0085-C03-01) and from the Comunidad Autónoma de Madrid (AE00022/94, 08.1/020/1997).

Publisher Summary DNA replication, gene transcription, and chromosome structure are regulated by proteins that bind specific DNA sequences. The DNA-binding specificities of many of these proteins have been characterized extensively in vitro. However, to determine the way they function in the context of a whole organism, the range of DNA sequences that they bind in vivo must be understood. This chapter describes a method that can directly quantitate binding by sequence-specific DNA binding proteins in vivo. There are three classes of in vivo DNA-binding assays: footprinting, immunolocalization, and cross-linking. The chapter describes these methods in detail and discusses the results obtained. Possible differences between transcription-factor DNA binding in prokaryotes and higher eukaryotes are also discussed. Footprinting assays provide the high-resolution mapping of DNA sequences that are protected from chemical or enzymatic attack by the binding of a protein. Immunolocalization of proteins on polytene chromosomes in Drosophila can identify the chromosomal regions at which proteins are found at their highest concentrations. Cross-linking assays employ either formaldehyde or ultraviolet (UV) light to covalently couple endogenous proteins to DNA. Although these methods have yielded important data, none can provide all of the information desired.

Mitochondrial RNA polymerase activity has been isolated from the crustacean Artemia franciscana at two stages of development, dormant embryo and developing larva. The preparations were obtained from purified mitochondria and the polymerase activity was purified by heparin-Sepharose chromatography. The presumed polymerase has a molecular mass of about 120 kDa and a 7.4S sedimentation coefficient. The biochemical characterization of the enzymatic reaction identified our RNA polymerase preparations as mitochondrial. The transcription initiation sites of Artemia mtDNA were characterized recently in our laboratory (J. A. Carrodeguas and C. G. Vallejo, Eur. J. Biochem. 250, 514-523, 1997). Artemia mtDNA fragments comprising the transcription initiation sites were transcribed by the partially purified polymerase preparation from the two developmental stages, but the transcription turned out to be unspecific. DNAse I footprinting analysis of a main transcription initiation site-containing DNA fragment revealed a protected region around the initiation site +1 position, when using a crude polymerase preparation. However, the protected region was not observed with the purified preparation. The results altogether suggest that a specificity factor is lost during purification. Based on the footprinting data, we suggest that the sequence from positions -6 to +13 of the main transcription initiation site in the Artemia mitochondrial DNA is the binding site of the homologous RNA polymerase holoenzyme., This work was supported by Research Grants PB89-0005 and PB92-0059 from the Dirección General de Investigación Científica y Técnica.

Publisher Summary This chapter discusses the genomic footprinting of budding yeast replication origins during the cell cycle. Nucleotide resolution genomic footprinting can be a powerful tool for the monitoring of protein-DNA interactions in vivo or in a cell extract. The technique involves the use of an enzymatic or chemical agent as a probe to induce modifications in the DNA as the first step. The presence of a protein bound to the DNA can alter the specificity of the probe, either reducing the level of modification (implying protection) or increasing the level of modification (resulting in hypersensitive sites). These alterations can include chemical or enzymatic cleavage of DNA or chemical modifications of the DNA. A number of genomic footprinting procedures have been described; however, the use of this technique has often been limited by problems encountered in the detection of signals derived from sequences that represent only a small fraction of the total DNA, such as single-copy sequences in the eukaryotic genome.

Publisher Summary This chapter summarizes the application and advantages of hydroxyl radicals for probing protein-binding sites on Ribonucleic acid (RNAs). Hydroxyl radicals are small, readily diffusible reagents that cleave ribose groups of the RNA backbone without preference for nucleotide or secondary structure character. Because of the small size of the hydroxyl radical reagent, the resulting footprinting data have much higher resolution than that offered by enzymatic cleavage methods. A stepwise approach for preparing the RNA transcripts, radiolabeling, protein binding, Fe(II)-ethylenediaminetetraacetic acid (EDTA) cleavage, and data analysis are provided; moreover, the advantages and experimental considerations relevant to the technique are described. Examples of hydroxyl radical footprinting are drawn from the analysis of RNA–coat protein interactions among the single-stranded, positive-sense RNA viruses, alfalfa mosaic virus, and the ilarviruses. The chapter discusses in detail about the prior uses of hydroxyl radical footprinting. The chapter also explains concepts related to preparing an RNA template for hydroxyl radical footprinting. Concepts of binding and cleavage reactions are also discussed. The chapter also presents an overview of footprinting data, and coupling hydroxyl radical footprinting with other methods.

Phage Φ29 protein p4 activates the late A3 promoter and represses the early A2c promoter, in both cases by binding upstream from RNA polymerase (RNAP) and interacting with the C-terminal domain of the RNAP α subunit. To investigate how this interaction leads to activation at P A3 and to repression at P A2c, mutant promoters were constructed. We show that the position of protein p4 relative to that of RNAP, which is different at each promoter, does not dictate the outcome of the interaction. Rather, in the absence of a −35 consensus box for σA-RNAP activation was observed, while in its presence repression occurred. The results support the view that stabilization of RNAP at the promoter over a threshold level leads to repression., This investigation has been aided by Research Grant 5R01 GM27242–18 from the National Institutes of Health, by Grant PB93–0173 from Dirección General de Investigación Científica y Técnica, by Grant CHRX-CT92–0010 from European Community, and by an Institutional Grant from Fundación Ramón Areces to the Centro de Biología Molecular “Severo Ochoa.” M. Monsalve and B. Calles were holders of predoctoral fellowships from Comunidad Autónoma de Madrid and Ministerio de Educación y Ciencia, respectively.

Publisher Summary This chapter describes a simple footprinting protocol, which yields reproducible, high-resolution genomic footprints from small amounts of mtDNA templates. This is generally applicable to study mitochondrial gene regulation in a wide variety of experimental samples where mitochondrial metabolism can be modified at will. Hence, genomic footprinting of mitochondrial DNA is an important tool in understanding mitochondrial gene expression. One important factor in obtaining useful footprint information is the uniformity of the cell or tissue sample. Heterogeneity in cell lineage or cell stage may lead to masking or overrepresentation of footprint data, leading to inaccurate conclusions. The advantage of this technique is that it is readily adaptable to other systems where the amount of DNA available does not warrant developing a litigation mediated polymerase chain reaction (LMPCR) protocol for genomic footprinting. This primer extension footprinting (PEF) protocol has been used to perform in vitro dimethyl sulfate (DMS) footprinting using recombinant plasmids and mitochondrial protein fractions.

The general inhibition in transcriptional activity during mitosis abolishes the stress-inducible expression of the human hsp70 gene. Among the four transcription factors that bind to the human hsp70 promoter, the DNA-binding activities of three (C/EBP, GBF, and HSF1) were normal, while Sp1 showed reduced binding activity in mitotic cell extracts. In vivo footprinting and immunocytochemical analyses revealed that all of the sequence-specific transcription factors were displaced from promoter sequences as well as from bulk chromatin during mitosis. The correlation of transcription factor displacement with chromatin condensation suggests an involvement of chromatin structure in mitotic repression. However, retention of DNase I hypersensitivity suggests that the hsp70 promoter was not organized in a canonical nucleosome structure in mitotic chromatin. Displacement of transcription factors from mitotic chromosomes could present another window in the cell cycle for resetting transcriptional programs. © 1995

Publisher Summary This chapter describes the protein-blotting procedures for evaluating the interactions of steroids receptors with DNA. Selective binding of steroid hormone receptors to hypoxia response element (HRE)-containing DNA fragments has been demonstrated by a variety of techniques for the study of DNA–protein interaction, including DNA–cellulose competitive binding, nitrocellulose filter binding, gel retardation, sucrose gradient shift, DNA footprinting (nuclease protection), and methylation interference assays, in addition to Southwestern blotting. Each of these techniques has its own set of advantages and limitations. Southwestern blotting, unlike nuclease protection or methylation interference studies, does not require the prior purification of the DNA-binding protein and can be used to study DNA–protein interactions in crude cellular extracts or in partially purified protein preparations at any stage of purification. Southwestern blotting offers further advantages in that once a protein extract has been electrophoretically separated and finally immobilized on a nitrocellulose filter, proteins of interest can potentially be characterized by a number of criteria, including relative mobility on the sodium dodecyl sulfate (SDS)-polyacrylamide gel, isoelectric point on two-dimensional gels, DNA-binding activity, ligand binding, or immunoreactivity, if antibodies for the protein are available.

Publisher Summary This chapter provides an overview on hydroxyl radical footprinting. Hydroxyl radical footprinting of DNA has evolved over the last several years into a facile and powerful technique for studying DNA structure and complexes of DNA with other molecules. The chapter describes the chemistry and history behind these techniques. In a footprinting experiment, a strand of DNA is subjected to cleavage by some reagent, both in the presence and absence of a DNA-binding ligand. Comparison of the cleavage patterns yields information on the structure of the DNA-ligand complex. Footprinting can also be used to obtain thermodynamic information on DNA-ligand complexes through footprint titration experiments.

Publisher Summary Growth, development, and differentiation of a cell involve important cellular processes such as DNA replication, recombination, and messenger RNA transcription that are controlled by specific interactions between nuclear proteins and defined DNA regulatory sequences. For instance, most transcription factors that regulate gene activity, either positively or negatively, are cell-specific or ubiquitous proteins that recognize and bind to their own promoter DNA motif, thereby influencing the function of the RNA polymerase at a variable distance in cis. Thus, a complex interplay between multiple DNA-binding proteins and their specific binding sites located within the promoter regions of eukaryotic genes is a fundamental determinant of both temporal regulation and tissue specificity of transcription. In order to resolve one specific protein-DNA complex from the many found in cells, the most successful strategy is to purify a DNA sequence and to use it to select from a mixture any protein that binds to it. Another approach is to use purified protein to select a specific DNA sequence. The method involves four steps: (1) separation of proteins by gel electrophoresis, (2) transfer of the separated proteins to a nitrocellulose filter, (3) detection of DNA-binding proteins by incubating the filter with a mixture of radioactively labeled DNA restriction fragments, and (4) identification of the DNA fragment bound to individual proteins by elution and gel electrophoresis. The advantage of this method is its high sensitivity and resolving power, allowing the direct screening of nuclear extracts.

Publisher Summary This chapter provides an overview on quantitative deoxyribonuclease I (DNase I) footprinting. DNase I footprinting was introduced by Schmitz and Galas to identify the DNA sequences that constitute binding sites for site-specific DNA-binding proteins. It has been used to detect changes in DNA structure and to measure the relative binding affinities of DNA-binding proteins. The unique value of footprinting as a quantitative titration technique for protein-DNA interactions is its ability to separately monitor the binding of protein to each specific site on the DNA. Further, the chapter focuses on the use of DNase I footprinting in vitro to yield data about specific binding sites and to obtain thermodynamic information that characterizes DNA-protein systems. One advantage of DNase I over chemical footprinting agents is that its enzymatic activity is specific to the DNA, so that degradation of the protein ligand is not a concern. A disadvantage is that its activity is somewhat sensitive to the DNA sequence, so that some regions are inefficiently cleaved even in the absence of bound protein ligand.

Publisher Summary This chapter provides an overview on hydroxyl radical footprinting. Hydroxyl radical footprinting of DNA has evolved over the last several years into a facile and powerful technique for studying DNA structure and complexes of DNA with other molecules. The chapter describes the chemistry and history behind these techniques. In a footprinting experiment, a strand of DNA is subjected to cleavage by some reagent, both in the presence and absence of a DNA-binding ligand. Comparison of the cleavage patterns yields information on the structure of the DNA-ligand complex. Footprinting can also be used to obtain thermodynamic information on DNA-ligand complexes through footprint titration experiments.

Publisher Summary The chapter describes the protein–DNA interactions in vivo by examining genes in Saccharomyces cerevisiae and Drosophila melanogaster by chromatin footprinting. The interactions of proteins with chromosomal DNA control a variety of cellular processes, including gene transcription, DNA packing, replication, recombination, and DNA repair. Specific cloned DNA sequences can be assembled with proteins isolated from cellular extracts to provide details on the binding of each factor and its effect on the overall structure and activity of the complex. One high-resolution method of mapping DNA–protein contacts that can be used with both reconstituted and cellular assemblies is DNA footprinting. This technique is designed to locate the binding sites of purified proteins on end-labeled DNA fragments by determining which DNA base pairs become selectively resistant to DNA cleavage reagents. The in vitro footprinting provides a high-resolution contact map with protein components that can be defined biochemically and assembled stepwise. The chromatin footprinting is most effective when used in parallel with biochemical studies as a predictive or confirmatory tool. The chapter discusses several viable alternatives and their strengths and weaknesses along with the protocols used to map DNase I sensitivity in Saccharomyces cerevisiae and methidium propyl-EDTA.Fe(II) sensitivity in Drosophila melanogaster .

Publisher Summary This chapter discusses the footprinting of protein–DNA complexes using γ rays. It presents an analog of the method of hydroxyl radical footprinting. The high-resolution footprints is duplicated of this technique, while substituting γ-radiation for the chemical reagents used heretofore. γ-Radiation has been shown to mediate DNA cleavage with no apparent base or sequence specificity. The primary cutting reagent, as in the analogous chemical reagent system, is thought to be the hydroxyl radical. This method requires no addition of reagents to the protein-DNA binding solution. The technique can be used to carry out footprinting in protein–DNA binding solutions that are not conducive to the use of the [Fe(II)EDTA] 2-reagent system, such as those that contain moderate amounts of glycerol or those that cannot tolerate the addition of one of the reagent components, such as hydrogen peroxide. Morever, the ability of γ-radiation to penetrate matter suggests that extension of the technique to in vivo systems might be possible. Such experiments could be performed on a model system constructed on a plasmid or could be applied to interactions of proteins with chromosomal DNA when used in combination with an appropriate detection scheme. An in vivo application would offer the opportunity of determining high-resolution footprints of proteins bound to DNA inside cells.