Mapping the interaction site of Rpb4 and Rpb7 subunits of RNA polymerase II in Saccharomyces cerevisiae

Rpb4 and Rpb7, the fourth and the seventh largest subunits of RNA polymerase II, form a heterodimer in Saccharomyces cerevisiae. To identify the site of interaction between these subunits, we constructed truncation mutants of both these proteins and carried out yeast two hybrid analysis. Deletions in the amino and carboxyl terminal domains of Rpb7 abolished its interaction with Rpb4. In comparison, deletion of up to 49 N-terminal amino acids of Rpb4 reduced its interaction with Rpb7. Complete abolishment of interaction between Rpb4 and Rpb7 occurred by truncation of 1-106, 1-142, 108-221, 172-221 or 198-221 amino acids of Rpb4. Use of the yeast two-hybrid analysis in conjunction with computational analysis of the recently reported crystal structure of Rpb4/Rpb7 sub-complex allowed us to identify regions previously not suspected to be involved in the functional interaction of these proteins. Taken together, our results have identified the regions that are involved in interaction between the Rpb4 and Rpb7 subunits of S. cerevisiae RNA polymerase II in vivo.     

Unstructured N terminus of the RNA polymerase II subunit Rpb4 contributes to the interaction of Rpb4.Rpb7 subcomplex with the core RNA polymerase II of Saccharomyces cerevisiae

Two subunits of eukaryotic RNA polymerase II, Rpb7 and Rpb4, form a subcomplex that has counterparts in RNA polymerases I and III. Although a medium resolution structure has been solved for the 12-subunit RNA polymerase II, the relative contributions of the contact regions between the subcomplex and the core polymerase and the consequences of disrupting them have not been studied in detail. We have identified mutations in the N-terminal ribonucleoprotein-like domain of Saccharomyces cerevisiae Rpb7 that affect its role in certain stress responses, such as growth at high temperature and sporulation. These mutations increase the dependence of Rpb7 on Rpb4 for interaction with the rest of the polymerase. Complementation analysis and RNA polymerase pulldown assays reveal that the Rpb4.Rbp7 subcomplex associates with the rest of the core RNA polymerase II through two crucial interaction points: one at the N-terminal ribonucleoprotein-like domain of Rpb7 and the other at the partially ordered N-terminal region of Rpb4. These findings are in agreement with the crystal structure of the 12-subunit polymerase. We show here that the weak interaction predicted for the N-terminal region of Rpb4 with Rpb2 in the crystal structure actually plays a significant role in interaction of the subcomplex with the core in vivo. Our mutant analysis also suggests that Rpb7 plays an essential role in the cell through its ability to interact with the rest of the polymerase.     

Mapping of Rpb3 and Rpb5 contact sites on two large subunits, Rpb1 and Rpb2, of the RNA polymerase II from fission yeast

[Rpb1 and Rpb2] Mapping of the contact sites␣on two large subunits of the fission yeast Schizosaccharomyces pombe RNA polymerase II with two small subunits, Rpb3 and Rpb5, was carried out using the two-hybrid screening system in the budding yeast Saccharomyces cerevisiae. Rpb5 was found to interact with any fragment of Rpb1 that contained the region H, which is conserved among the subunit 1 homologues of all RNA polymerases, including the β' subunit of prokaryotic RNA polymerases. In agreement with the fact that Rpb5 is shared among all three forms of eukaryotic RNA polymerases, the region H of RNA polymerase I subunit 1 (Rpa190) was also found to interact with Rpb5. On the other hand, two-hybrid screening of Rpb2 fragments from RNA polymerase II indicated the presence of an Rpb3 contact site in the region H which is conserved among the subunit 2 homologues of all RNA polymerases, including the β subunit of prokaryotic RNA polymerases. Possible functions of the regions H in the subunits 1 and 2 are discussed. Received: 10 December 1997 / Accepted: 14 April 1998     

Rsp5 Ubiquitin-Protein Ligase Mediates DNA Damage-Induced Degradation of the Large Subunit of RNA Polymerase II in Saccharomyces cerevisiae

Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 proteins. We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation of SEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW domain-containing hect (WW-hect) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.