Transcription of eukaryotic genes by RNA polymerase II (Pol II) is typically accompanied by nucleosome survival and minimal exchange of histones H3/H4. The mechanism of survival and recovery of chromatin structure remains obscure. Here we show how transcription through chromatin by Pol II is uniquely coupled with nucleosome survival. Structural modeling and functional analysis of the intermediates of transcription through a nucleosome was conducted. When Pol II approaches the area of strong DNA-histone interactions, a small intranucleosomal DNA loop (zero-size or Ø-loop) containing transcribing enzyme is formed. During formation of the Ø-loop, the recovery of DNA-histone interactions behind Pol II is tightly coupled with their disruption ahead of the enzyme. This coupling is a distinct feature of the Pol II-type mechanism that allows further transcription through the nucleosome, prevents nucleosome translocation and minimizes displacement of H3/H4 histones from DNA during enzyme passage.
Single positioned nucleosomes have been extensively employed as simple model experimental systems for analysis of various intranuclear processes. Here we describe an experimental system containing positioned mononucleosomes allowing transcription by various RNA polymerases. Each DNA template contains a pair of fluorescent labels (Cy3 and Cy5) allowing measuring relative distances between the neighboring coils of nucleosomal DNA using Forster resonance energy transfer (FRET). The single-particle FRET (spFRET) approach for analysis of DNA uncoiling from the histone octamer during transcription through chromatin is described in detail.
Aerogels with a density of 4.2−22.8 kg/m 3 were obtained from bacterial cellulose synthesized under static and dynamic cultivation conditions on a molasses medium. The strength properties and porous structure of the aerogels strongly depended on their density. With an aerogel density of 22.8 kg/m 3 , the modulus of elasticity at 80% compression of the sample was 0.1 MPa. The decrease in the density of aerogels led to an increase in the pore sizes ranging from 20 to 1000 μm and a decrease in the modulus of elasticity. These characteristics were more pronounced in aerogels obtained from bacterial cellulose under static cultivation conditions. The aerogels had a low coefficient of thermal conductivity (0.0257 W m −1 °C−1 ), which is comparable to the thermal conductivity of air, and moderate thermal stability because the degradation processes of the aerogels began at 237 °C. The aerogels obtained from bacterial cellulose had high sound absorption coefficients in the frequency range of 200−5000 Hz, which makes it possible to use the aerogels as heat-and sound-insulating materials.
Purified fractions of cytosol, vacuoles, nuclei, and mitochondria of Saccharomyces cerevisiae possessed inorganic polyphosphates with chain lengths characteristic of each individual compartment. The most part (80-90%) of the total polyphosphate level was found in the cytosol fractions. Inactivation of a PPX1 gene encoding ~40-kDa exopolyphosphatase substantially decreased exopolyphosphatase activities only in the cytosol and soluble mitochondrial fraction, the compartments where PPX1 activity was localized. This inactivation slightly increased the levels of polyphosphates in the cytosol and vacuoles and had no effect on polyphosphate chain lengths in all compartments. Exopolyphosphatase activities in all yeast compartments under study critically depended on the PPN1 gene encoding an endopolyphosphatase. In the single PPN1 mutant, a considerable decrease of exopolyphosphatase activity was observed in all the compartments under study. Inactivation of PPN1 decreased the polyphosphate level in the cytosol 1.4-fold and increased it 2- and 2.5-fold in mitochondria and vacuoles, respectively. This inactivation was accompanied by polyphosphate chain elongation. In nuclei, this mutation had no effect on polyphosphate level and chain length as compared with the parent strain CRY. In the double mutant of PPX1 and PPN1, no exopolyphosphatase activity was detected in the cytosol, nuclei, and mitochondria and further elongation of polyphosphates was observed in all compartments.
Isolated mitochondria of Saccharomyces cerevisiae cells grown on glucose possess acid-soluble inorganic polyphosphate (polyP). Its level strongly depends on phosphate (P(i)) concentration in the culture medium. The polyP level in mitochondria showed 11-fold decrease under 0.8 mM P(i) as compared with 19.3 mM P(i). When spheroplasts isolated from P(i)-starved cells were incubated in the P(i)-complete medium, they accumulated polyP and exhibited a phosphate overplus effect. Under phosphate overplus the polyP level in mitochondria was two times higher than in the complete medium without preliminary P(i) starvation. The average chain length of polyP in mitochondria was of <15 phosphate residues at 19.3 mM P(i) in the culture medium and increased at phosphate overplus. Deoxyglucose inhibited polyP accumulation in spheroplasts, but had no effect on polyP accumulation in mitochondria. Uncouplers (FCCP, dinitrophenol) and ionophores (monensin, nigericin) inhibited polyP accumulation in mitochondria more efficiently than in spheroplasts. Fast hydrolysis of polyP was observed after sonication of isolated mitochondria. Probably, the accumulation of polyP in mitochondria depended on the proton-motive force of their membranes.
Mutants of Saccharomyces cerevisiae with inactivated endopolyphosphatase gene PPN1 did not grow on lactate and ethanol, and stopped growth on glucose earlier than the parent strain. Their mitochondria were defective in respiration functions and in metabolism of inorganic polyphosphates. The PPN1 mutants lacked exopolyphosphatase activity and possessed a double level of inorganic polyphosphates in mitochondria. The average chain length of mitochondrial polyphosphates at the stationary growth stage on glucose was about 15-20 and about 130-180 phosphate residues in the parent strain and PPN1 mutants, respectively. Inactivation of the PPX1 gene encoding exopolyphosphatase had no effect on respiration functions and on polyphosphate level and chain length in mitochondria.
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