Activity of purified and chromatin-associated mouse TLT hepatoma RNA polymerases on homologous whole chromatin and euchromatin-enriched segments isolated from homologous chromatin

Abstract

The RNA polymerase (EC 2.7.7.6) activity of mouse TLT hepatoma nuclei is separable into three forms, I, IIA, and IIB; each of these partially purified enzymes demonstrates characteristics generally similar to those reported for these enzymes of other systems. All three forms of TLT hepatoma RNA polymerase show a considerable preference for single-stranded DNA. The full expression of the endogenous RNA polymerase activity of mouse TLT hepatoma chromatin is dependent on the salt concentration. No additional template activity to added RNA polymerase I or II is available. Physical shearing decreased endogenous RNA polymerase activity and increased template capacity to the added enzymes. Glycerol-gradient fractionation of physically sheared chromatin gave a fairly diffuse distribution of the endogenous RNA polymerase activity to the marginally euchromatin-enriched fractions. However, enzymatic shearing of TLT hepatoma chromatin by Mg,Ca-dependent autodigestion results in a distribution of endogenous RNA polymerase activity to the highly euchromatin-enriched fractions similar to that obtained for nascent RNA (Paul, I. J. &Duerksen, J. D. (1976) Biochem. Biophys. Res. Commun. 68, 97–105). The distribution patterns of template capacity determined with added RNA polymerase I and II differed somewhat from the above distributions and varied with the length of autodigestion. Shearing by autodigestion is preferred, and followed by glycerol-gradient centrifugation permits a considerable enrichment for euchromatic segments.