Molecular assembly of RNA polymerase II from the fission yeast Schizosaccharomyces pombe: subunit-subunit contact network involving Rpb5.
Eukaryotic RNA polymerase II is composed of more than 10 polypeptide chains. The minimum and essential subunits for RNA synthesis have not yet been identified. Toward this ultimate goal, we analysed the topological arrangement of the putative subunits. Here we report a subunit-subunit contact network involving subunit 5 of the fission yeast Schizosaccharomyces pombe RNA polymerase II.
The rpb5+ gene encoding subunit 5 of RNA polymerase II was cloned from the fission yeast Schizosaccharomyces pombe. The polypeptide predicted from DNA sequence of the rpb5+ gene consists of 210 amino acids with a calculated molecular weight of 23914. The homology of the amino acid sequence is 55% and 43% with Saccharomyces cerevisiae RPB5 and human hRPB25, respectively. Far-Western blot analysis of S. pombe RNA polymerase II using 32P-labelled recombinant Rpb5 fused to glutathione S-transferase (GST) as a probe, indicated that Rpb5 binds strongly to membrane-immobilized Rpb1, Rpb2 and Rpb3 and weakly to Rpb5 and a 15-kDa subunit (Rpb8 or Rpb11). In agreement with this result, the 32P-labelled Rpb3 probe showed a strong binding signal against Rpb5 in addition to Rpb1 and Rpb2. The existence of Rpb5-Rpb3 contact was supported by detection of complexes formed between these two proteins synthesized in vitro using protein-immobilized beads.
Rpb3 and Rpb5, the putative subunits of RNA polymerase II, associate each other to form binary complexes. These two subunits also bind to the two large subunits, Rpb1 and Rpb2, independently.
Miyao T, Yasui K, Sakurai H, Yamagishi M, Ishihama A