Involvement of multiple subunit-subunit contacts in the assembly of RNA polymerase II.
RNA polymerase II from the fission yeast Schizo-saccharomyces pombe consists of 12 species of subunits, Rpb1-Rpb12. We expressed these subunits, except Rpb4, simultaneously in cultured insect cells with baculovirus expression vectors. For the isolation of subunit complexes formed in the virus-infected cells, a glutathione S -transferase (GST) sequence was fused to the rpb3 cDNA to produce GST-Rpb3 fusion protein and a decahistidine-tag sequence was inserted into the rpb1 cDNA to produce Rpb1H protein. After successive affinity chromatography on glutathione and Ni(2+)columns, complexes consisting of the seven subunits, Rpb1H, Rpb2, GST-Rpb3, Rpb5, Rpb7, Rpb8 and Rpb11, were identified. Omission of the GST-Rpb3 expression resulted in reduced assembly of the Rpb11 into the complex. Direct interaction between Rpb3 and the other six subunits was detected by pairwise coexpression experiments. Coexpression of various combinations of a few subunits revealed that Rpb11 enhances Rpb3-Rpb8 interaction and consequently Rpb8 enhances Rpb1-Rpb3 interaction to some extent. We propose a mechanism in which the assembly of RNA poly-merase II is stabilized through multiple subunit-subunit contacts.
Kimura
M
M
Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan. makimura@lab.nig.ac.jp
Ishihama
A
A
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D011485:Protein Binding; D012319:RNA Polymerase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012568:Schizosaccharomyces / Q000201:enzymology; D018411:Spodoptera
D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D012319:RNA Polymerase II