Abstract
Class III DNA-dependent RNA polymerases were purified from the mouse plasmacytoma, MOPC 315. RNA polymerases IIIA and IIIB were solubilized from a whole cell extract and resolved by chromatography on DEAE-Sephadex. Chromatography on DEAE-cellulose, DEAE-Sephadex, CM-Sephadex, and phosphocellulose ion exchange resins and sedimentation in sucrose density gradients yielded chromatographically homogeneous Enzymes IIIA and IIIB which were purified approximately 22,000 and 53,000-fold respectively, relative to whole cell extracts. The specific activity of these enzymes was comparable to that reported for other purified eukaryotic RNA polymerases. Sucrose gradient sedimentation analysis suggested a molecular weight of approximately 650,000 for each of the class III enzymes.
Highlights
The subunit compositions of chromatographically purified RNA polymerases III, and III, were analyzed by polyacrylamide gel electrophoresis under denaturing conditions
The cellular levels of solubilized RNA polymerase III activity vary among different cell types and in the same cell type under different physiological conditions [2,3,4] and may regulate directly the cellular rate of tRNA and -5 S RNA synthesis
This paper reports the purification and subunit structures of the class III enzymes from MOPC 315 cells, which have permitted a structural comparison of homologous class I, II, and III RNA polymerases [16]
Summary
GM-70661 from the National Institutes of Health. polymerase III usually accounts for a small proportion of the total RNA polymerase activity and has been detected, using appropriate analytical methods, in all tissues examined [2]. Determination of the specific mechanism(s) accounting for these variations in RNA polymerase III activity might provide insights into the regulation of tRNA and 5 S RNA synthesis and cell growth rates. To investigate these problems, we have chosen the mouse plasmacytoma, MOPC 315,2 a rapidly growing malignant cell. Evidence is presented that the heterogeneous class III enzymes, designated III, and III., have minor differences in their physical properties and subunit compositions These studies provide evidence that fluctuations in the levels of RNA polymerase III activity may, in part, be mediated via changes in enzyme concentration
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