Involvement of multiple subunit-subunit contacts in the assembly of RNA polymerase II

RNA polymerase II from the fission yeast Schizosaccharomyces 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-infecte d cells, a glutathione S-transferase (GST) sequence was fused to the rpb3 c DNA to produce GST-Rpb3 fusion protein and a decahistidine-tag sequence was inserted into the rpb1 cDNA to produce Rpb1H protein, After successive aff inity chromatography on glutathione and Ni2+ columns, complexes consisting of the seven subunits, Rpb1H, Rpb2, GST-Rpb3, Rpb5, Rpb7, Rpb8 and Rpb11, w ere identified. Omission of the GST-Rpb3 expression resulted in reduced ass embly of the Rpb11 into the complex. Direct interaction between Rpb3 and th e other six subunits was detected by pairwise coexpression experiments. Coe xpression of various combinations of a few subunits revealed that Rpb11 enh ances Rpb3-Rpb8 interaction and consequently Rpb8 enhances Rpb1-Rpb3 intera ction to some extent, We propose a mechanism in which the assembly of RNA p olymerase II is stabilized through multiple subunit-subunit contacts.