Abstract
We have used affinity chromatography on columns containing immobilized calf thymus RNA polymerase II to isolate three phosphoproteins (RAP72, RAP38, and RAP30) that bind directly to RNA polymerase II. All could be isolated from cell nuclei, and all three could be detected in mouse and human tissue culture cell lines, but only RAP38 and RAP30 have so far been isolated from calf thymus. RAP38 stimulates nonspecific transcription of native DNA templates by RNA polymerase II in the presence of Mn2+; it appears to be similar or identical to SII, a previously identified RNA polymerase II stimulatory factor (Nakanishi, Y., Mitsuhashi, Y., Sekimizu, K., Yokoi, H., Tanaka, Y., Horikoshi, M., and Natori, S. (1981) FEBS Lett. 130, 69-72). Unlike RAP38, RAP72 and RAP30 do not affect nonspecific transcription by RNA polymerase II. However, RAP30 may have a role in regulating some alterations of transcription that accompany cellular differentiation; RAP30 is partially dephosphorylated when murine erythroleukemia cells are induced with dimethyl sulfoxide to undergo terminal erythroid differentiation. We suggest that phosphate groups in RNA polymerase II-binding proteins may regulate transcription by modulating the interaction of RNA polymerase II with other regulatory proteins that possess sequence recognition specificity.
Highlights
Protein-coding genes in eukaryotic cells are transcribed by DNA-dependent RNA polymerase I1
Detection of RNA Polymerase 11-bindingProteins-Whole cell extracts were prepared asdescribed under "Materials and Methods." Each extract was applied to a micro-affinity column containingimmobilized calf thymus RNApolymerase I1 and to a control column containingno covalentlycoupled protein ligand
The calf thymus RNA polymerase I1 used here for affinity chromatography was always at least 95% pure, but identical results have been obtainedwith 70-80% pure enzyme not subjected to the final purification step[24]
Summary
Detection of RNA Polymerase 11-bindingProteins-Whole cell extracts were prepared asdescribed under "Materials and Methods." Each extract was applied to a micro-affinity column containingimmobilized calf thymus RNApolymerase I1 and to a control column containingno covalentlycoupled protein ligand. Increased approximately linearly with the amount of extract loaded (Fig. 1B) It was, necessary to load a 20-pl column with at least IO7 cell eq, corresponding to approximately 50mg of cell extract protein/ml of column bed, in order to reliably detect the RNA polymerase 11-bindingproteins above the background. SDS-PAGE mobilities as those found in MELC were found in mouse Y1 adrenocortical tumor cells and in human HeLa cells (Fig. 3) It is, likelythat RAP72, RAP38, and RAP30 are found in most or all types of mouse and humantissueculture cell lines and are not tissue-specific proteins. A parallel experiment done with MELC did detect RAP72 (Fig. 3) It is, possible that RAP72 is found only in column and which could be dissociated from the columns with tumor cells or only in cells grown in tissue culture. Types-RNA polymerase 11-binding proteins with the same RAP72,RAP38, and RAP30 CanBe Isolated from Cell
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