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14. Nucleolar DNA-Dependent RNA Polymerase from Rat Liver 1. Purification and Subunit Structure.

Author

Matsui, Takashi, Onishi, Toshio, and Muramatsu, Masami

Subjects
*DNA, *RNA, *POLYMERASE chain reaction, *RATS, *GENES, *ENZYMES
Abstract

DNA-dependent RNA polymerase I (or A) was purified from rat liver nucleoli. DNA was effectively removed from the solubilized enzyme with a defined concentration of polyethyleneglycol. The enzyme was purified further with successive DEAE-Sephadex and phosphocellulose column chromatography followed by glycerol gradient centrifugation. The procedure yielded an electro- phoretically homogeneous enzyme with a specific activity 400 times that of the nucleolar extracts. The recovery of the activity was approximately 20%. The RNA polymerase I eluted as a single peak from DEAE-Sephadex was separated into two distinct peaks by a phosphocellulose column. The first peak eluting at about 0.12 M ammonium sulfate was designated as RNA polymerase IA and the second peak eluting at about 0.18 M as RNA polymerase IB. In normal rat liver nucleoli IA enzyme comprised approximately 20 %, of the total RNA polymerase I activity and the JB enzyme comprised approximately 80%. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, enzyme IB contained five subunits with molecular weights of 195000 (a), 130000 (b), 65000 (c). 40000 (d) and 19000 (e) at nearly equimolar amounts. The calculated molecular weight of the enzyme (449000) agreed well with that predicted from the sedimentation coefficient of the enzyme Enzyme IA contained identical subunits except that subunit c was absent. Preliminary studies could not demonstrate any significant differences in template specificity between IA and IB enzyme. [ABSTRACT FROM AUTHOR]

Published
1976
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15. Nucleolar DNA-Dependent RNA Polymerase from Rat Liver 1. Two Forms and Their Physiological Significance.

Author

Matsui, Takashi, Onishi, Toshio, and Muramatsu, Masami

Subjects
*RNA polymerases, *DNA, *ENZYMES, *PHYSIOLOGY, *ACTINOMYCIN, *HEPATECTOMY
Abstract

RNA polymerase I (or A) was extracted from nuclear. nucleolar and nucleoplasmic fractions. and resolved into IA and IB forms on a phosphocellulose column, During the course of cycloheximide treatment, the activity of RNA polymerase IB decreased in the nucleoli with concomitant increase in the nucleoplasmic fraction, suggesting strongly that cycloheximide induced specific leakage of IA enzym from the nucleolus. The activity of IA enzyme did not change in the nucleoli. When nucleoli were incubated in the presence of actinomycin D, all the IA enzyme activity and approximately 30% of the total IB enzyme activity were released in the incubation medium, where 70%, of IB activity remained associated with the nucleolar pellet where no IA, activity was detected. The enzyme which was released into the incubation medium was tentatively designated as free or unbound RNA polymerase I and that which was associated with the nucleolar pellet as template-bound enzyme. During the treatment with cycloheximide, the activity of bound enzyme, which contained exclusively IB form, decreased rapidly, with kinetics almost identical to that of nucleolar RNA synthesis in vitro. The activity of free enzyme did not change appreciably. At 2 h after partial hepatectomy. IB enzyme activity in the free RNA polymerase fraction increased to almost twice the control, while the bound enzyme activity did not increase appreciably until 4 h of regeneration. Enhancement of nucleolar RNA synthesis in viva was not apparent at 2 h but became significant by 4 h after partial hepatectomy. These results strongly suggest that (a) the above-mentioned procedure is actually fractionating RNA polymerase I into free and bound forms, (b) RNA polymerase IB is the transcriptionally active form in viva. (c) RNA polymerase IB exists in excess in the nucleoli, but the amount of bound IB molecules, which are engaged in transcription in vivo, must be determined by some other factor(s) than the mere concentration of IB enzyme in the nucleolus, and (d) IA form is not an artifact of isolation hut is always present vi vivo at a certain amount, although the exact nature of this molecule is not known at present. [ABSTRACT FROM AUTHOR]

Published
1976
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