Study of the loosely bound non-histone chromatin proteins. Stimulation of deoxyribonucleic acid-templated ribonucleic acid synthesis by a specific deoxyribonucleic acid-binding phosphoprotein fraction

Abstract

The loosely bound chromatin proteins of Ehrlich ascites hyperdiploid cells have been prepared by extraction of chromatin with 0.35 M NaCl. Sodium dodecyl sulfate gel electrophoresis of the 0.35 M NaCl-soluble chromatin proteins reveals high heterogeneity with a molecular weight range of 10,000 to 170,000. The 0.35 M NaCl-soluble chromatin proteins contain many components similar to the more tightly bound non-histone chromatin proteins complex with the loosely bound chromatin proteins by gradient dialysis, the inhibitory effect of histones on transcription of DNA in vitro was reduced. The reconstituted complex manifested a level of template activity similar to that of native chromatin as measured in an Ehrlich ascites tumor RNA polymerase reaction. The loosely bound chromatin proteins contain RNA as well as phosphoproteins. Phenol extraction or DNA affinity chromatography of these proteins yielded fractions enhanced 25- to 30-fold in phosphorus which were capable of stimulating DNA-templated RNA synthesis in vitro. The stimulation of transcription from DNA was template-specific, effective only with a DNA template prepared from Ehrlich ascites tumor, but not from rat liver, calf thymus, or chicken erythrocytes. In addition, the stimulatory effect of the specific DNA-binding proteins appears to be RNA polymerase-specific, the stimulation being manifested with Ehrlich ascites tumor nucleoplasmic RNA polymerase and not with Micrococcus luteus RNA polymerase. Thus, the loosely bound chromosomal proteins from Ehrlich ascites tumor contain a fraction that specifically binds to Ehrlich ascites tumor DNA and exhibits a template- and RNA polymerase-specific stimulatory effect on transcription from DNA.