Template Properties of Rat Liver Chromatin after Restricted Protein Supply; Transcription by Homologous Ribonucleic Acid Polymerase I and II

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Abstract
Rats were fed a diet containing either 20% or 3% of 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 using pancreatic ribonuclease A activity as a reference standard. Endogenous ribonuclease activity was less than 10 ng/mg chromatin-DNA and there were no significant differences between the two groups. The DNA:RNA:protein ratio of the chromatin was 1:0.125:2.84 and 1:0.145:2.48 for rats fed 20% and 3% protein, respectively. The differences were not significant. Template efficiency of the chromatin was measured using DNA-dependent RNA polymerase I or II prepared from liver of rats fed a stock diet. Under ionic conditions optimal for the two enzymes, the chromatin-dependent incorporation of precursor [3H]UTP into RNA continued for 40 minutes at 35°. Differences in template activity between the two dietary groups became apparent after separation of the RNA product on sucrose density gradient or by polyacrylamide gel electrophoresis. The major 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 two 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.