|
内容記述 |
The subunit-subunit contact network within the Schizosaccharomyces pombe RNA polymerase II was analyzed using various methods. Far-Western blot analysis of pairs of over-expressed and isolated subunits using six small-sized subunits, Rpb6, Rpb7, Rpb8, Rpb10, Rpb11 and Rpb12, as probes indicated the subunit-subunit interaction for a total of 18 (or 19) combinations. Taken together with our previous analyses, the eight small-sized subunits, Rpb3, Rpb5, Rpb6, Rpb8, Rpb1O, Rpb11 and Rpb12, were identified to bind one or both of two large subunits, Rpb1 and Rpb2. In addition, the bimolecular interaction was observed for the combination of Rpb3-Rpb11. The subunit-subunit contact within the assembled RNA polymerase was then analyzed by protein-protein cross-linking using five molecular species of the bifunctional cross-linker with different linker length and specificity. Cross-linking was observed for a total of 19 combinations, including five combinations between small subunits, Rpb3-Rpb1O. 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 besides small subunits interact each other for limited combinations. Direct contact of the two large subunits, Rpb1 and Rpb2, was also demonstrated by cross-linking.<br /> Since Rpb6 was found to make multiple contacts with not only the two large subunits but also three small subunits Rpb5, Rpb7 and Rpb8, genetic analysis was carried out for the common subunit Rpb6 shared by RNA polymerases I, II and III. Deletion and truncation anayses of the rpb6 gene indicated that Rpb6 consisting of 142 amino acid residues is an essential protein for cell viability, but the essential region is located in the carboxy (C)-terｍinal proximal half between residues 61-139. After random mutagenesis, a total of 14 temperature-sensitive (Ts) mutants were isolated, each carrying a single (or double in three cases and triple in one case) mutation. Four mutants each carrying a amino acid change in the essential region were sensitive against 6-azauracil (6AU) that inhibits transcription elongation by depleting the intracellular pool of GTP and UTP. Both 6AU sensitivity and Ts phenotypes of these rpb6 mutants were suppressed by over-expression of TFIIS, a transcription elongation factor. In agreement with the genetic studies, the direct interaction between TFIIS and RNA polymerase II was observed in vitro by pull-down assay using a fusion form of TFIIS with glutathione S-tansferase (GST), and the mutant RNA polymerases containing the mutant Rpb6 subunits showed reduced affinity for TFIIS. Taken together we propose that Rpb6 plays a central role in protein-protein assembly with both the RNA polymerase II subunits and the transcriptiion elongation factor TFIIS. |
要旨・審査要旨 / Abstract, Screening Result (209.1 kB)
