Transcription of fractionated calf thymus chromatin by RNA polymerase of calf thymus and Escherichia coli

Abstract

Calf thymus chromatin has been sheared and fractionated on sucrose gradients. Approximately 5-10% of the chromatin is resolved from the bulk of the imput chromatin as a slowly sedimenting fraction. The protein/DNA ratio of the slowly sedimenting fraction is not greatly different from the protein/DNA ratio of the more rapidly sedimenting chromatin fraction. Analysis of DNA of the chromatin fractions by CsC1 equilibrium density gradient centrifugation indicates that DNA of the slowly sedimenting fraction is depleted in the satellite DNA banding at 1.716 g/cm3. The template properties of the chromatin fractions have been examined with Escherichia coli RNA polymerase and form II and form III RNA polymerases of calf thymus. At rate-limiting concentrations, the slowly sedimenting fraction is twofold more active than the rapidly sedimenting fraction as a template for E. coli RNA polymerase. Homologous form II and form III RNA polymerases are respectively 30-fold and 16-fold more active with the slowly sedimenting fraction than the rapidly sedimenting fraction. The activity of form II RNA polymerase in transcribing the slowly sedimenting fraction exceeds its activity in transcribing an equal concentration of native DNA. Kinetic studies, in which RNA polymerase activity is assayed at various concentrations of chromatin, indicate that the greater activity of E. coli RNA polymerase with the slowly sedimenting fraction is due to an increased rate of transcription at saturating concentrations of template (Vmax), and is not due to a lower concentration required for half-maximal rate of transcription (Km). In contrast, the increased rates of transcription of the slowly sedimenting chromatin faction by the homologous polymerases are due to a decrease in concentration required for half-maximal rate of transcription rather than an increased rate of transscription at saturation concentrations of template. The relative degrease of satellite DNA in the slowly sedimenting fraction of chromatin and the enhanced template activity of the slowly sedimenting fraction suggest that this fraction is equivalent to nuclear euchromatin while the more rapidly sedimenting chromatin is equivalent to nuclear heterochromatin.

Transcription of isolated mouse liver chromatin

“…As these experiments measure the accessibility of DNA sequences within chromatin for transcription and not the accuracy of initiation differences in the frequency of transcription of individual RNA species in vivo and in vitro might result from the use of a heterologous polymerase. Using fractionated calf thymus chromatin, Henner et al (1975) have shown that in most cases the rate of RNA synthesis with E. coli RNA polymerase is greater than the rate with homologous RNA polymerase. The increase in rate of RNA synthesis could reflect a change in the frequency with which "correct" sequences are transcribed or an increase in the number of sequences being transcribed.…”

Composition and template activity of chromatin fractionated by isoelectric focusing

“…After washing 4 times with 10 mL of 10% trichloroacetic acid in 40 mM sodium pyrophosphate, the filter was incubated wtih 1 mL of Protosol (NEN) for 10 h, and then 10 drops of acetic acid and 15 mL of Triton-toluene (Austin et al, 1973) scintillation fluid were added. RNA polymerase preparations were obtained as described by Henner et al (1975).…”

“…Paul, 1969) have made it increasingly desirable to separate transcriptionally active chromatin from total chromatin. Fractionation has been attempted by a variety of techniques including mechanical breakage (Arnold and Young, 1974;Chesterton et al, 1974;Clark and Felsenfeld, 1971) or nuclease digestion (Gottesfeld et al, 1974) followed by fractionation techniques based on differences in size (Janowski et al, 1972), density (Frenster et al, 1963;McCarthy et al, 1974;Henner et al, 1975), and charge (Simpson, 1974).…”

Enzymic polyadenylation of 5S ribosomal ribonucleic acid and synthesis of a complementary deoxyribonucleic acid

“…The homogenate was filtered through cheesecloth and centrifuged at 500g for 15 min (4 °C) to pellet the nuclei. The nuclei were then homogenized as above in 100 mL of 0.3 M sucrose containing 10 mM Tris-Cl (pH 8.0), 3 mM CaCl2, 5 mM magnesium acetate, 5 mM dithiothreitol, 0.1% Triton X-100 (Henner et al, 1975), heparin, dextran sulfate (20 gg/mL), and poly(vinyl sulfate) (20 µg/mL•) to remove ribosomes adhering to the endoplasmic reticulum associated with the nucleus. The washed nuclei and postnuclear supernatant were (separately) centrifuged at 18000g for 25 min.…”