Two large subunits of the fission yeast RNA polymerase II provide platforms for the assembly of small subunits.

The subunit-subunit contact network was analyzed for the Schizosaccharomyces pombe RNA polymerase II consisting of ten putative subunits. Previously we carried out far-Western blot analysis of bimolecular interaction with radio-labeled subunit 3 and 5 probes. Here we extended the analysis using another six small-sized subunits as probes. Taking the results together the subunit-subunit interaction was observed for a total 18 (or 19) combinations. All eight small-sized subunits exhibited binding activities to two large subunits, Rpb1 and Rpb2. In addition, bimolecular interaction was observed for the combinations of Rpb3-Rpb5, Rpb3-Rpb11 (and Rpb5-Rpb8/11). The subunit-subunit contact within the assembled RNA polymerase was then analyzed by protein-protein cross-linking using five species of bifunctional cross-linkers with different length and specificity. Cross-linking was observed for a total of 19 combinations, including five combinations between small subunits, Rpb3-Rpb10, Rpb3-Rpb11, Rpb5-Rpb6, Rpb6-Rpb7 and Rpb6-Rpb8. The results altogether indicate that two large subunits Rpb1 and Rpb2 provide the platform for assembly of small subunits and also small subunits interact with each other for limited combinations. Direct contact of the two large subunits, Rpb1 and Rpb2, was also demonstrated by cross-linking.

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