Phosphorylation of the RNA Polymerase II Carboxy-Terminal Domain by the Bur1 Cyclin-Dependent Kinase

Abstract

BUR1, which was previously identified by a selection for mutations that have general effects on transcription in Saccharomyces cerevisiae, encodes a cyclin-dependent kinase that is essential for viability, but none of its substrates have been identified to date. Using an unbiased biochemical approach, we have identified the carboxy-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II, as a Bur1 substrate. Phosphorylation of Rpb1 by Bur1 is likely to be physiologically relevant, since bur1 mutations interact genetically with rpb1 CTD truncations and with mutations in other genes involved in CTD function. Several genetic interactions are presented, implying a role for Bur1 during transcriptional elongation. These results identify Bur1 as a fourth S. cerevisiae CTD kinase and provide striking functional similarities between Bur1 and metazoan P-TEFb.The largest subunit of RNA polymerase II (Pol II), Rpb1, contains a highly conserved carboxy-terminal domain (CTD) that has a central role in transcriptional regulation in vivo (3, 11). The Rpb1 CTD consists of multiple repeats of the consensus heptapeptide sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser, which is repeated 26 times in Saccharomyces cerevisiae, 42 times in Drosophila melanogaster, and 52 times in humans and mice (9). Although the CTD is not required for RNA polymerase activity in promoter-independent assays, it is essential in vivo; deletion of the entire CTD in Drosophila and S. cerevisiae results in lethality, while truncation to 11 repeats in yeast confers conditional growth and promoter-specific transcriptional defects (36).Phosphorylation of the CTD is important for regulation of Pol II activity during the transcription cycle: unphosphorylated Pol II is preferentially recruited into the preinitiation complex (PIC) (33) and then becomes phosphorylated during the transition from initiation to elongation (28). CTD phosphorylation thus has both stimulatory and inhibitory roles; phosphorylation prior to PIC assembly inhibits initiation, while phosphorylation after PIC assembly stimulates promoter escape and elongation. Phosphorylation occurs primarily on serine 2 and serine 5 of the consensus CTD repeat, with serine 2-phosphorylated Rpb1 being enriched distally from the promoter and serine 5-phosphorylated Rpb1 being enriched at promoter-proximal regions (26). Hyperphosphorylation of the CTD is also linked to other essential events during mRNA synthesis, including recruitment of mRNA modification enzymes and pre-mRNA splicing factors (reviewed in reference 50).The importance of CTD phosphorylation for Pol II regulation has prompted efforts to identify the kinases and phosphatases that determine the CTD phosphorylation state. Several kinases capable of phosphorylating the CTD in vitro have been identified in Drosophila, human, and rodent cell extracts (reviewed in reference 11), but it is not clear whether they all function as CTD kinases in vivo. In S. cerevisiae, where sophisticated genetic analysis can be readily combined with biochemistry to de...

Interactions between Fission Yeast Cdk9, Its Cyclin Partner Pch1, and mRNA Capping Enzyme Pct1 Suggest an Elongation Checkpoint for mRNA Quality Control

“…The amino acid sequence of SpCdk9 is most closely related to the sequences of the HsCdk9 (372 amino acids) and DmCdk9 (401 amino acids) subunits of human and Drosophila P-TEFb (21) and to the essential S. cerevisiae cyclin-dependent kinase Bur1 (657 amino acids), which phosphorylates the pol II CTD and is the putative functional equivalent of Cdk9 in budding yeast (18). SpCdk9 and Bur1 are 200 -250 amino acids larger than the metazoan Cdk9 proteins; this difference is attributable to the presence of Cterminal domains in the fungal Cdk9 proteins that have no counterpart in HsCdk9 and DmCdk9 (Fig.…”

“…A C-terminal domain of S. pombe Cdk9, separate from the kinase domain, suffices for binding to Pct1 in vivo and in vitro. We identify Pch1 as the cyclin partner of SpCdk9 and show genetically that SpCdk9/Pch1 are the functional orthologs of the S. cerevisiae CTD kinase Bur1/Bur2, a putative fungal counterpart of P-TEFb (18). Based on these findings, we invoke an Spt5-induced elongation checkpoint, the purpose of which is to recruit the capping enzymes, in which P-TEFbmediated release from the elongation block may be facilitated by contacts between P-TEFb and a component of the capping apparatus.…”

Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

“…Although both have been shown to assemble into preinitiation complexes (PICs) at the promoter, recent experiments utilizing analogue-sensitive mutants of both kinases suggest different, but partially overlapping roles for both enzymes in promoting transcription (12). The other two CTD kinases, Ctk1 in CTDK-I and Sgv1/Bur1, have been associated primarily with the elongation phase of transcription (13)(14)(15)(16)(17). Biochemical studies indicate that CTDK-I can stimulate elongation (17), and in vivo experiments show that elongating Pol II is aberrantly phosphorylated in ctk1 mutants (16,18).…”

Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 Protein Kinase

“…The mammalian pol II CTD has 52 heptad repeats, the fission yeast Schizosaccharomyces pombe CTD has 29 repeats, and the budding yeast Saccharomyces cerevisiae CTD has 26 copies. The pol II CTD undergoes a cycle of extensive phosphorylation and dephosphorylation at positions Ser 5 and Ser 2 , which is coordinated with the transcription cycle (3). Multiple CTD kinases are present in eukaryotic cells, e.g.…”

“…The essential Bur1 Cdk of budding yeast and its cyclin partner Bur2 are putative orthologs of the Cdk9 and cyclin T subunits of the metazoan protein kinase P-TEFb (4,5). P-TEFb is a transcription elongation factor that overrides the negative actions of Spt5 and its associated factors.…”