A simple solubilization method for loosely and tightly chromatin-bound RNA polymerase II from rat liver nuclei.
Rat liver chromatin-bound RNA polymerase II could be differentially solubilized into two distinct populations, loosely and tightly bound enzymes, by a simple method. By this method the recovery of the solubilized enzyme from the chromatin fraction could be increased considerably as compared with the procedure of Yu (1). The two chromatin-bound enzymes had different properties: (a) Loosely bound enzyme was easily extractable from chromatin with relatively mild ionic condition (0.5 M NaCl); the tightly bound enzyme had to be solubilized by more drastic conditions such as sonication or nuclease treatment. (b) Loosely bound enzyme could not efficiently transcribe the chromatin template, but the tightly bound enzyme was active toward the same template. The latter enzyme is involved in the tight complex with the RNA synthesis activating factors. (c) Cycloheximide treatment in vivo suggests that the two enzymes have different turn-over rates. Therefore, with this simple solubilization method the functionally different two chromatin-bound RNA polymerase II activities can be estimated.
D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology*; D002843:Chromatin / Q000201:enzymology*; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology*; D008297:Male; D011485:Protein Binding; D012319:RNA Polymerase II / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
D002843:Chromatin; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases