Template properties of rat liver chromatin after restricted protein supply; trascription by homologous ribonucleic acid polymerase I and II
von der Decken, A.; Andersson, G.M.
Journal of Nutrition 107(6): 949-958
1977
ISSN/ISBN: 0022-3166 PMID: 864523 Document Number: 120024
Rats were fed on a diet containing either 20 or 3% high-quality protein. After 6 days, liver chromatin was prepared by extraction of the nuclei with Tris buffer-NaCl-EDTA. Endogenous RNA polymerase was inactivated by incubation with N-ethylmaleimide. Endogenous ribonuclease activity was estimated with pancreatic ribonuclease A activity as a reference standard. Endogenous ribonuclease activity was less than 10 ng/mg chromatin-DNA and there was no significant difference between the 2 groups. The DNA:RNA:protein ratio of the chromatin was 1:0.125:2.84 and 1:0.145:2.48 for rats given 20 and 3% protein, respectively. The differences were not significant. Template efficiency of the chromatin was measured with DNA-dependent RNA polymerase I or II prepared from liver of rats fed on a stock diet. Under ionic conditions best suited for the 2 enzymes, the chromatin-dependent incorporation of precursor [3H]UTP into RNA continued for 40 min at 35 deg . Differences in template activity between the 2 dietary groups became apparent after separation of the RNA product on sucrose density gradient or by polyacrylamide gel electrophoresis. The main part of the radioactive RNA was in the range of 4S to 18S. Kinetic analysis indicated that the template concentration required for half maximum velocity was similar for the chromatin of the 2 dietary groups, but the enzyme RNA polymerase I needed twice the amount of chromatin required by RNA polymerase II to obtain half maximum velocity of RNA synthesis.