Extensive chromatin fragmentation improves enrichment of protein binding sites in chromatin immunoprecipitation experiments

Extensive sonication of formaldehyde-crosslinked chromatin can generate DNA fragments averaging 200 bp in length (range 75–300 bp). Fragmentation is largely random with respect to genomic region and nucleosome position. ChIP experiments employing such extensively fragmented samples show 2- to 4-fold increased enrichment of protein binding sites over control genomic regions, when compared to samples sonicated to a more conventional size range (300–500 bp). The basis of improved fold enrichments is that immunoprecipitation of protein-bound regions is unaffected by fragment size, whereas immunoprecipitation of control genomic regions decreases progressively along with reduced fragment size due to fewer nonspecific binding sites. The use of extensively sonicated samples improves mapping of protein binding sites, and it extends the dynamic range for quantitative measurements of histone density. We show that many yeast promoter regions are virtually devoid of histones.     

Homologous RNA polymerase binding to Escherichia coli DNA: a study of the distribution of stable binding sites.

The number and the distribution of the sites of Escherichia coli DNA that form stable complexes with the homologous RNA polymerase (class A sites according to Hinkle and Chamberlin [3]) have been investigated. Almost all the DNA can bind RNA polymerase, even when fragmented at short (undergenic) size; this general non-promoter-specific binding is highly labile and is not temperature-dependent. The range of RNA polymerase/DNA ratios that give rise to the stable temperature-dependent complexes was examined. The amount and the distribution of class A complexes were studied analysing the dissociation of complexes formed by RNA polymerase on DNA fragments of various length. The E. coli genome appears to form 3.8 X 10(3) stable complexes; the majority of these complexes shows a short-range distribution (800-1200 base pairs). The rest is more widely spaced (1200-6000 base pairs).     

Initiation of RNA molecules by purified Escherichia coli RNA polymerase

Summary The kinesties of appearance of, and the distribution among, the four bases of the chain-initial nucleotides (5-termini) of RNA chains synthesized in vitro under various conditions have been investigated. The results of this study have shown that when native T4 bacteriophage DNA is the template for the RNA polymerase, most chains start with a purine nucleotide. The ratio of ATP termini to GTP termini is independent of the reaction time and of the template/enzyme ratio in the reaction mixture. A similar preferential purine initiation was observed when denatured T4 is the template, but the ratio of ATP to GTP termini is reduced. All 5-termini of poly-AU chains synthesized on poly-dAT templates are ATP.The determination of the kinetics of initiation of RNA chains has allowed direct confirmation of some conclusions which had been inferred previously from sedimentation analyses of the RNA product. (1) Most RNA chains are initiated during a short period at the outset of the reaction. (2) Low concentrations of native DNA templates limit the number of RNA chains synthesized, not the rate of RNA chain growth. (3) The maximum number of RNA molecules which can be synthesized on denatured DNA templates is severalfold larger than the maximum number which can be synthesized on the same weight of native DNA.     

On the binding of tRNA to Escherichia coli RNA polymerase.

The fixation of tRNA to Escherichia coli RNA polymerase has been investigated. Bound and free tRNA have been separated and quantified after filtration through cellulose nitrate filters, centrifugation or sucrose gradients or electrophoresis in polyacrylamide gels. We detect no differences between the fixation of E. coli fMet-tRNAfMet, Met-tRNAmMet or uncharged unfractionated tRNA to RNA polymerase. Tight complexes, with a long residence time, are formed between core enzyme and tRNA with a dissociation constant of less than 1 nM. Complexes exist between tRNA and both monomer and dimer forms of the core enzyme. In the monomer complex, one tRNA is bound per alpha 2 beta beta' unit, whereas in the dimer complex only 0.5 tRNA molecule is fixed per alpha 2 beta beta' unit. In contrast to the core enzyme, very little tRNA fixes tightly to the holoenzyme at salt concentrations greater than 80 mM. At lower salt concentrations tRNA fixation results in a loss of sigma subunit from the holo enzyme to the resulting core enzyme where it binds tightly. DNA fixation reduces the binding of tRNA to RNA polymerase and tRNA fixation reduces the binding of DNA. However, binding of DNA to polymerase is not competitive with binding of tRNA, and ternary complexes between RNA polymerase, DNA and tRNA are shown to exist. Our results are discussed in relation to other studies concerning the effects of tRNA upon RNA polymerase.     

Localization of Escherichia coli RNA polymerase binding sites on bacteriophage S13 and 0X174 DNAs by electron microscopy.

Complexes between Escherichia coli RNA polymerase and bacteriophage S13 and phage phiX174 replicative form III DNAs have been shown to form at specific locations on the phage genomes. The major locations on S13 have been mapped at 8 to 10 and 92 to 96% of the genome length, starting from the unique Pst I cleavage site. The locations correspond to the beginnings of genes D and B, respectively. Four minor locations map at 18 to 22, 28 to 32, 50 to 56, and 70 to 74% of the genome. The 70 to 74% site corresponds to the beginning of the A gene. The major locations on phiX174 are at 8 to 10, 50 to 54, and 92 to 94% of the genome. The 50 to 54% site is at the start of the H gene and has an equivalent minor site on S13, but it is not a promoter site. Three minor sites on phiX174, at 20 to 24, 26 to 32, and 68 to 74% of the genome, correspond to sites on S13. The data confirm the locations of sites identified by restriction fragment binding experiments (E. Rassart and J. H. Spencer, J. Virol. 27:677--687, 1978) and the assignment of putative promoters at the start of genes A, B and D.     

The structure of the zinc sites of Escherichia coli DNA-dependent RNA polymerase.

X-ray absorption spectroscopy is ideally suited for the investigation of the electronic structure and the local environment (approximately 5 A) of specific atoms in biomolecules. While the edge region provides information about the valence state of the absorbing atom, the chemical identity of neighboring atoms, and the coordination geometry, the extended x-ray absorption fine structure region contains information about the number and average distance of neighboring atoms and their relative disorder. The development of sensitive detection methods has allowed studies using near physiological concentrations (as low as approximately 100 microM). RNA polymerase from Escherichia coli contains two zinc atoms: one tightly bound in the beta' subunit, the subunit that participates in template binding, and the other loosely bound in the beta subunit, the subunit that participates in substrate binding. X-ray absorption studies of these zinc sites in the native protein and of the zinc site in the beta' subunit after removal of the zinc in the beta subunit site by p-(hydroxymercuri)benzenesulfonate (Giedroc, D. P., and Coleman, J. E. (1986) Biochemistry 25, 4969-4978) indicate that both zinc sites have octahedral coordination. The zinc in the beta' subunit site has four sulfur ligands at an average distance of 2.36 +/- 0.02 A and two oxygen (or nitrogen) ligands at an average distance of 2.23 +/- 0.02 A. The beta subunit zinc site has five sulfur ligands at an average distance of 2.38 +/- 0.01 A and one histidine nitrogen ligand at 2.14 +/- 0.02 A. These results are in general agreement with earlier biochemical and spectroscopic studies.