Intracellular contents and assembly states of all 12 subunits of the RNA polymerase II in the fission yeast Schizosaccharomyces pombe.
The RNA polymerase II (Pol II) of the fission yeast Schizosaccharomyces pombe is composed of 12 different polypeptides, Rpb1 to Rpb12, of which five, Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12, are shared among three forms of the RNA polymerase. To get an insight into the control of synthesis and assembly of individual subunits, we have measured the intracellular concentrations of all 12 subunits in S. pombe by quantitative immunoblotting. Results indicate that the levels are low for the three large subunits, Rpb1, Rpb2 and Rpb3, which are the homologues of beta', beta and alpha subunits, respectively, of prokaryotic RNA polymerase. On the other hand, the levels of small-sized subunits were between 2- to 15-fold higher than these three core subunits. The levels of the five common subunits shared among RNA polymerases I, II and III are about 10 times greater than those of the Pol II-specific core subunits. The assembly state of the Rpb proteins was analyzed by glycerol gradient centrifugation of S. pombe whole cell extracts. The three core subunits are mostly assembled in Pol II, but some of the small subunits were detected in the slowly sedimenting fractions, indicating that at least some of the excess Rpb proteins exist in unassembled forms. Based on the intracellular concentration of the least abundant Rpb3 subunit, the total number of Pol II in a growing S. pombe cell was estimated to be about 10,000 molecules. The intracellular distribution of some Pol II subunits was also analyzed by microscopic observation of the green fluorescent protein (GFP)-fused Rpb proteins. In agreement with the biochemical analysis, the GFP-Rpb1 and GFP-Rpb3 fusions were present in the nuclei but the GFP-Rpb4 was detected in the cytoplasm as well as the nuclei.
Kimura
M
M
National Institute of Genetics, Department of Molecular Genetics, Mishima, Shizuoka, Japan.
Sakurai
H
H
Ishihama
A
A
D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005990:Glycerol / Q000378:metabolism; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D012318:RNA Polymerase I / Q000737:chemistry; D012319:RNA Polymerase II / Q000737:chemistry* / Q000378:metabolism; D012320:RNA Polymerase III / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins
D008164:Luminescent Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D049452:Green Fluorescent Proteins; D012319:RNA Polymerase II; C112110:RPB3 protein, S cerevisiae; C491782:rpb3 protein, S pombe; D012318:RNA Polymerase I; D012320:RNA Polymerase III; D005990:Glycerol