Composition and template activity of chromatin fractionated by isoelectric focusing

HeLa cell interphase chromatin has been sheared and fractionated by isoelectric focusing. Chromatin fractions are obtained with a wide range of isoelectric points. No free DNA is observed. While protein/DNA rations are similar in the various fractions, they appear to contain different nonhistone chromosomal proteins. A minor chromatin fraction with isoelectric point congruent to 7.0 does not contain histone H1. This fraction is considerably more active as template with different RNA polymerases than the other fractions. Kinetic studies, in which RNA polymerase activity is assayed at various concentrations of chromatin, indicate that the greater activity of Escherichia coli RNA polymerase 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 transciption (Km). In contrast, the increased rate of transcription by calf-thymus RNA polymerases II and III is due to a decrease in chromatin concentration required for half-maximal rate of transcription rather than an increased rate of transcription at saturating concentrations of template. These results suggest that chromatin with isoelectric point congruent to 7 offers a greater frequency of binding sites for mammalian RNA polymerases, as would be expected for a "transcriptionally active" fraction.