Rsp5 WW domains interact directly with the carboxyl-terminal domain of RNA polymerase II

RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to form a physical and functional complex with RNA polymerase II (RNA pol II). The amino-terminal half of Rsp5 consists of four domains: a C2 domain, which binds membrane phospholipids; and three WW domains, which are protein interaction modules that bind proline-rich ligands. The carboxyl-terminal half of Rsp5 contains a HECT (homologous to E6-AP carboxyl terminus) domain that catalytically ligates ubiquitin to proteins and functionally classifies Rsp5 as an E3 ubiquitin-protein ligase. The C2 and WW domains are presumed to act as membrane localization and substrate recognition modules, respectively. We report that the second (and possibly third) Rsp5 WW domain mediates binding to the carboxyl-terminal domain (CTD) of the RNA pol II large subunit. The CTD comprises a heptamer (YSPTSPS) repeated 26 times and a PXY core that is critical for interaction with a specific group of WW domains. An analysis of synthetic peptides revealed a minimal CTD sequence that is sufficient to bind to the second Rsp5 WW domain (Rsp5 WW2) in vitro and in yeast two-hybrid assays. Furthermore, we found that specific "imperfect" CTD repeats can form a complex with Rsp5 WW2. In addition, we have shown that phosphorylation of this minimal CTD sequence on serine, threonine and tyrosine residues acts as a negative regulator of the Rsp5 WW2-CTD interaction. In view of the recent data pertaining to phosphorylation-driven interactions between the RNA pol II CTD and the WW domain of Ess1/Pin1, we suggest that CTD dephosphorylation may be a prerequisite for targeted RNA pol II degradation.     

The role of TFIIB-RNA polymerase II interaction in start site selection in yeast cells

Previous studies have established a critical role of both TFIIB and RNA polymerase II (RNAPII) in start site selection in the yeast Saccharomyces cerevisiae. However, it remains unclear how the TFIIB–RNAPII interaction impacts on this process since such an interaction can potentially influence both preinitiation complex (PIC) stability and conformation. In this study, we further investigate the role of TFIIB in start site selection by characterizing our newly generated TFIIB mutants, two of which exhibit a novel upstream shift of start sites in vivo. We took advantage of an artificial recruitment system in which an RNAPII holoenzyme component is covalently linked to a DNA-binding domain for more direct and stable recruitment. We show that TFIIB mutations can exert their effects on start site selection in such an artificial recruitment system even though it has a relaxed requirement for TFIIB. We further show that these TFIIB mutants have normal affinity for RNAPII and do not alter the promoter melting/scanning step. Finally, we show that overexpressing the genetically isolated TFIIB mutant E62K, which has a reduced affinity for RNAPII, can correct its start site selection defect. We discuss a model in which the TFIIB–RNAPII interaction controls the start site selection process by influencing the conformation of PIC prior to or during PIC assembly, as opposed to PIC stability.     

The splicing factor, Prp40, binds the phosphorylated carboxyl-terminal domain of RNA polymerase II

We showed previously that the WW domain of the prolyl isomerase, Ess1, can bind the phosphorylated carboxyl-terminal domain (phospho-CTD) of the largest subunit of RNA Polymerase II. Analysis of phospho-CTD binding by four other WW domain-containing Saccharomyces cerevisiae proteins indicates the splicing factor, Prp40, and the RNA polymerase II ubiquitin ligase, Rsp5, can also bind the phospho-CTD. The identification of Prp40 as a phospho-CTD binding protein represents the first demonstration of direct interaction between a documented splicing factor and the phospho-CTD. Domain dissection studies reveal that phospho-CTD binding occurs at multiple locations in Prp40, including sites in both the WW and FF domain regions. Because the conserved repeats of the CTD make it an ideal ligand for multi-site binding events, the implications of multi-site binding are discussed. Our data suggest a mechanism by which the phospho-CTD of elongating RNA polymerase II facilitates commitment complex formation by juxtaposing the 5' and 3' splice sites.