Interaction of Fcp1 Phosphatase with Elongating RNA Polymerase II Holoenzyme, Enzymatic Mechanism of Action, and Genetic Interaction with Elongator

Abstract

Fcp1 de-phosphorylates the RNA polymerase II (RNAPII) C-terminal domain (CTD) in vitro, and mutation of the yeast FCP1 gene results in global transcription defects and increased CTD phosphorylation levels in vivo. Here we show that the Fcp1 protein associates with elongating RNAPII holoenzyme in vitro. Our data suggest that the association of Fcp1 with elongating polymerase results in CTD de-phosphorylation when the native ternary RNAPII0-DNA-RNA complex is disrupted. Surprisingly, highly purified yeast Fcp1 dephosphorylates serine 5 but not serine 2 of the RNAPII CTD repeat. Only free RNAPII0(Ser-5) and not RNA-PII0-DNA-RNA ternary complexes act as a good substrate in the Fcp1 CTD de-phosphorylation reaction. In contrast, TFIIH CTD kinase has a pronounced preference for RNAPII incorporated into a ternary complex. Interestingly, the Fcp1 reaction mechanism appears to entail phosphoryl transfer from RNAPII0 directly to Fcp1. Elongator fails to affect the phosphatase activity of Fcp1 in vitro, but genetic evidence points to a functional overlap between Elongator and Fcp1 in vivo. Genetic interactions between Elongator and a number of other transcription factors are also reported. Together, these results shed new light on mechanisms that drive the transcription cycle and point to a role for Fcp1 in the recycling of RNAPII after dissociation from active genes. During the RNA polymerase II (RNAPII)1 transcription cycle, both the phosphorylation state of the polymerase and the proteins that RNAPII interacts with change dramatically. Throughout promoter recruitment and initiation where RNA-PII exists in a hypo-phosphorylated state, it interacts with general transcription factors, such as TFIID, TFIIB, TFIIF, TFIIE, TFIIH, and Mediator (1). In the process of promoter clearance and early transcript elongation where RNAPII becomes hyper-phosphorylated, interactions with initiation-specific factors are severed, and interactions with other proteins are established. Factors that have been shown to preferentially associate with hyper-phosphorylated elongating RNAPII include RNA processing factors (2) and factors thought to play a role in transcript elongation, such as Elongator, Set1, and Set2 (3-8).The C-terminal repeat domain (CTD) of RNAPII is the specific target for kinases that phosphorylates the polymerase at the transition from initiation to elongation. The CTD consists of the conserved heptapeptide, Tyr-Ser-Pro-Thr-Ser-Pro-Ser, repeated 26 times in yeast and up to 52 times in metazoans (9). After transcript initiation, the CTD is heavily phosphorylated on serine 2 and serine 5 by different protein kinases (pTEFb (yeast Bur1 or Ctdk1)) and TFIIH), respectively (10, 11). Later, during transcript elongation and at transcriptional termination, CTD phosphatases remove the phosphate groups, enabling recycling of RNAPII for a new round of transcription (11,12). A CTD phosphatase was first identified biochemically through its ability to specifically dephosphorylate the RNAPII CTD in vitro (13). The gene encoding a TFIIF-inter...

Identification of Genes Affecting Hydrogen Sulfide Formation inSaccharomyces cerevisiae

“…The role of this gene in sulfide formation is unclear since it, like ATP11, is predicted to affect multiple cellular processes. Hht2p has been reported to be associated with elongator, an RNA polymerase II-associated histone acetylase that facilitates transcription, and several other factors in a complex (23). Two other proteins identified in our study that affect sulfide production, Sit4p and Iki3p, are also known to form a complex with Hht2p (15,23,37).…”

“…Hht2p has been reported to be associated with elongator, an RNA polymerase II-associated histone acetylase that facilitates transcription, and several other factors in a complex (23). Two other proteins identified in our study that affect sulfide production, Sit4p and Iki3p, are also known to form a complex with Hht2p (15,23,37). SIT4 is involved in transcriptional regulation (9), and IKI3 encodes for a subunit of the elongator complex (7,22,23).…”

“…Two other proteins identified in our study that affect sulfide production, Sit4p and Iki3p, are also known to form a complex with Hht2p (15,23,37). SIT4 is involved in transcriptional regulation (9), and IKI3 encodes for a subunit of the elongator complex (7,22,23). The fact that deletion of all three of these genes leads to increased H 2 S suggests that the reported interaction of their gene products is of biological significance and this complex exerts some form of regulation on the reduction of sulfide, either directly or indirectly.…”

Fcp1 directly recognizes the C-terminal domain (CTD) and interacts with a site on RNA polymerase II distinct from the CTD

“…This realization reemphasizes the issue of what factors͞conditions are responsible for rendering the CTD of elongating Pol II refractory to Fcp1 phosphatase (53). It is necessary to further characterize Fcp1 interaction with Pol II ternary complexes engaged at various stages of transcription elongation as opposed to the unengaged Pol II because conflicting results have been reported in this regard (39,53,54).…”

“…Although it is known that transcription-specific kinases and phosphatases are the main players (46,52,53), how exactly the dynamics of CTD phosphorylation are determined by their actions throughout the transcription cycle remains unclear. Actions by CTD phosphatases such as Fcp1 must be pivotal in this transaction, given the known functions (39,46).…”