RNA polymerase II subunits 2, 3, and 11 form a core subassembly with DNA binding activity.
RNA polymerase II purified from the fission yeast Schizosaccharomyces pombe consists of 10 species of subunit polypeptide. We introduced a histidine cluster tag sequence into the chromosomal rpb1 and rpb3 genes, which encode subunit 1 (Rpb1) and subunit 3 (Rpb3), respectively, and purified the RNA polymerase by Ni2+ affinity chromatography. After stepwise dissociation of the Rpb1- and Rpb3-tagged RNA polymerases fixed on Ni2+-resin by increasing concentrations of urea or guanidium hydrochloride, Rpb2-Rpb3-Rpb11 or Rpb2-Rpb3-Rpb11-Rpb10 complexes were obtained. Since the complex consisting of Rpb2, Rpb3, and Rpb11 cannot be dissociated even after treatment with 6 M urea buffer, we propose that this complex represents a core subassembly of the RNA polymerase II, analogous to the alpha2beta complex in the assembly of Escherichia coli RNA polymerase. Both the Rpb2-Rpb3-Rpb11 complex and the free Rpb1 protein showed DNA binding activity, although the affinity was weaker compared with the intact RNA polymerase.
Kimura
M
M
Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411, Japan.
Ishiguro
A
A
Ishihama
A
A
D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism*; D010957:Plasmids; D011487:Protein Conformation; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D012568:Schizosaccharomyces / Q000201:enzymology
D004247:DNA; D012319:RNA Polymerase II