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.The carboxyl-terminal domain (CTD) 1 of the largest subunit of RNA polymerase II (1) plays a central role in mRNA synthesis. The CTD is composed of 26 -52 heptad repeats with the consensus sequence, YSPTSPS. These repeats are extensively phosphorylated in RNA polymerase II actively engaged in transcript elongation (2-5).In the past several years evidence has accumulated indicating that the phosphorylated form of the CTD acts to coordinate pre-mRNA processing events (for review see Refs. 6 -10). The 5Ј capping complex is not only localized near the pre-mRNA by direct association with the phospho-CTD (11, 12) but is also allosterically activated by phospho-CTD binding (13,14). A variety of evidence strongly suggests that the phospho-CTD is involved in splicing. The hyperphosphorylated form of RNA polymerase II colocalizes with splicing factors when transcriptionally active (15, 16) and in the nuclear matrix (17-19). Several phospho-CTD binding proteins have been identified that contain SR and RRM domains as found in many splicing factors (20,21). Fusion proteins with a hyperphosphorylated CTD can inhibit splicing in vivo (22). In addition, transcriptionally unengaged but phosphorylated RNA polymerase II is able to stimulate splicing in vitro (23).Recently, we demonstrated that the prolyl isomerase, Ess1, can bind the phosphorylated form of the CTD and that this binding is mediated by its WW domain (24). Because the prolyl isomerase activity of Ess1 preferentially acts on phospho-SerPro peptide bonds, as are found in abundance in the phospho-CTD, our findings provided a plausible explanation for earlier results implicating Ess1 in pre-mRNA 3Ј end formation (25,26).WW domains, named for two highly conserved tryptophan residues, are small independently folding protein domains consisting of slightly more than 30 amino acids arranged in three anti-parallel ␤ sheets (27, 28). These domains have been shown to bind proline-rich sequences containing several different motifs (29). Of five protei...

A phospho-carboxyl-terminal domain (CTD) affinity column created with yeast CTD kinase I and the CTD of RNA polymerase II was used to identify Ess1/Pin1 as a phospho-CTD-binding protein. Ess1/Pin1 is a peptidyl prolyl isomerase involved in both mitotic regulation and pre-mRNA 3-end formation. Like native Ess1, a GSTEss1 fusion protein associates specifically with the phosphorylated but not with the unphosphorylated CTD. Further, hyperphosphorylated RNA polymerase II appears to be the dominant Ess1 binding protein in total yeast extracts. We demonstrate that phospho-CTD binding is mediated by the small WW domain of Ess1 rather than the isomerase domain. These findings suggest a mechanism in which the WW domain binds the phosphorylated CTD of elongating RNA polymerase II and the isomerase domain reconfigures the CTD though isomerization of proline residues perhaps by a processive mechanism. This process may be linked to a variety of pre-mRNA maturation events that use the phosphorylated CTD, including the coupled processes of pre-mRNA 3-end formation and transcription termination.The carboxyl-terminal domain (CTD) 1 of the largest subunit of RNA polymerase II plays a central role in controlling mRNA production in eukaryotes. In most eukaryotes the CTD consists of between 26 and 52 repeats with the consensus sequence, YSPTSPS (1). This unusual domain is not found in eukaryotic RNA Pol I or Pol III or in prokaryotic RNA polymerase. By mechanisms that are still largely not understood, RNA Pol II behavior is controlled by CTD kinases and phosphatases that modulate the phosphorylation state of the CTD (2). A body of evidence has accumulated indicating that the unphosphorylated CTD is present in RNA Pol II holoenzyme and is required during the initial stages of transcription (3-6) but that a hyperphosphorylated CTD is present in elongating polymerase (7-10).The hypothesis that the phospho-CTD functions in pre-mRNA processing (11) has received firm experimental backing with the recognition that several components of the 5Ј mRNA capping enzyme complex are phospho-CTD-binding proteins (12)(13)(14). In addition, indirect evidence suggests that the phospho-CTD also binds splicing components (15-18). Further, both the phosphorylated and the unphosphorylated CTD can interact with pre-mRNA 3Ј-end processing proteins (19,20). The combined evidence suggests that pre-mRNA processing may in general be cotranscriptional, with the (phospho)-CTD serving as a master coordinator of the events involved.Recently, mutations in the peptidyl prolyl isomerase, ESS1/ PTF1, were found to be responsible for the temperature-sensitive phenotypes of two yeast mutants that displayed a pre-mRNA 3Ј-end formation defect (21). Yeast ESS1 was originally identified as essential gene (22), and is in fact the only essential peptidyl prolyl isomerase (PPIase) in Saccharomyces cerevisiae (23). The human protein, Pin1, and the Drosophila protein, Dodo, are close homologues of Ess1 and both can functionally replace Ess1 in yeast (24, 25). All three proteins h...

Mass spectrometry has been used to demonstrate that vitamin K-dependent carboxylation is a processive posttranslational modification (i.e. multiple carboxylations occur during a single association between enzyme and substrate). Purified vitamin K-dependent carboxylase can carboxylate as many as 12 glutamate residues in FIXQ/S, a peptide substrate based on amino acids ؊18 to 41 of the human blood clotting enzyme factor IX. Mass spectrometry was used to determine the number of ␥-carboxyl groups added to FIXQ/S by the carboxylase during an in vitro reaction. Despite the fact that most substrate molecules in a reaction were uncarboxylated, almost all carboxylated FIXQ/S molecules were carboxylated many times. This observation can only be explained by two types of mechanisms. In a processive mechanism, multiple carboxylations could occur during a single substrate binding event. Alternatively, a distributive mechanism could result in the observed behavior if the initial carboxylation event results in a substrate that is additionally carboxylated far more efficiently than the uncarboxylated FIXQ/S. Kinetic experiments and arguments were used to show that the vitamin K-dependent carboxylase is not distributive but rather is one of the first well documented examples of an enzyme that catalyzes a processive post-translational modification.