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

Abstract

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 Rpb4⅐Rbp7 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.Studies of transcriptional regulation have focused mainly on the role of DNA-bound regulatory proteins and their contacts with the general transcription factors, mediator, and other accessory proteins in the transcriptional machinery. In most eukaryotes, the 12-subunit RNA polymerase II (Pol II) 4 is thought to have little or no influence on the regulation of transcription. Rpb4 and Rpb7 form a subcomplex within the polymerase that dissociates easily under mild denaturing or nondenaturing conditions and shows variable association with the polymerase at different growth stages, leading to the suggestion that the subcomplex could be analogous to the subunit of the bacterial RNA polymerase (1-3). Whether such a regulatory role can be ascribed to the subcomplex has been a matter of some debate, but the phenotypes of the deletion mutants and the interactions mediated by these subunits suggest that they might have some regulatory role in stress response and transcription (4).Rpb7 is essential for survival of Saccharomyces cerevisiae, whereas Rpb4 is not (5). However, rpb4⌬ strains are temperature-sensitive and cold-sensitive, show poor recovery from stationary phase, are defective in sporulation (a response to severe nutritional starvation), and are predisposed to pseudohyphae formation (a response to mild nutritional starvation) (4). Apart from its roles in stress response, Rpb4 is involved in transcription under moderate and extreme temperatures (6). The polymerase lacking Rpb4 and Rpb7 is defective for promoter-dependent initiation of transcription but not for promoter-inde...

Mediator Head module structure and functional interactions

“…Notably, the subunits are essential for promoter-dependent initiation but not for elongation29, and they appear to be involved in a step of initiation subsequent to recruitment of RNAPII to the PIC30. Recent analysis suggests that physical interaction with RNAPII underlies the essential role of Rpb7 in the cell31. The results presented here suggest that this might at least in part be due to the critical role Rpb4 and Rpb7 play in enabling interaction of Mediator with RNAPII.…”

“…It has been suggested21,22 that interaction of the N terminus of Rpb7 with the switch domains at the base of the RNAPII clamp would prevent the latter from adopting a more open conformation that would be necessary to accommodate double-stranded DNA in the RNAPII active site cleft. Rather than blocking clamp movement, the interaction of Rpb4 and Rpb7 (the N terminus of Rpb4 also interacts with the clamp31,32) with the clamp might allow the subunits to function as a ‘handle’ that could facilitate clamp movement in response to interaction of RNAPII with Mediator and other components of the transcription machinery (Fig. 5b).…”

A genome wide dosage suppressor network reveals genomic robustness

“…Six mutants were ts lethal due to missense mutation ( med4, med11, tfb3, rad3, kin28, taf12 ); two were ts deletion mutants ( rpb4Δ and taf14Δ ). The deletion mutants had complete deletion of the respective structural genes, such that there was no possibility of expression of any remnant protein fragment (49,64). …”

“…These observations are generally consistent with the recent observation that RNA Pol II open complexes can be reactivated by the TFIIE complex through stabilizing effects on relatively unstructured domains on mediator proteins (74). Such ‘Intrinsically Disordered Regions’ serve to functionally assemble RNA pol II complex subunit proteins (64,75) and thus provide a high degree of modular functionality.…”

RNA Polymerase II Phosphorylation and Gene Expression Regulation

“…Crystal structures of the Rpb4/7 heterodimer in the context of the complete RNAPII complex localized it in the proximity of the Rpb1-CTD [203,207], and biochemical and genetic studies suggest that Rpb4/7 might have a function in the recruitment of some CTD-binding proteins to transcribing RNAPII. Moreover, it is possible that this sub-complex, Rpb4/7, would regulate the access of CTD modifying enzymes during the whole transcription cycle [203,207,[209][210][211][212]. Actually, structural studies have provided further evidence that the CTD extends from the RNAPII core enzyme near the RNA exit channel [204], where it is ideally located to bind and be affected by the action of a multitude of factors, among them kinases, phosphatases and isomerases.…”