CTD phosphatase: role in RNA polymerase II cycling and the regulation of transcript elongation.

The repetitive C-terminal domain (CTD) of the largest RNA polymerase II subunit plays a critical role in the regulation of gene expression. The activity of the CTD is dependent on its state of phosphorylation. A variety of CTD kinases act on RNA polymerase II at specific steps in the transcription cycle and preferentially phosphorylate distinct positions within the CTD consensus repeat. A single CTD phosphatase has been identified and characterized that in concert with CTD kinases establishes the level of CTD phosphorylation. The involvement of CTD phosphatase in controlling the progression of RNAP II around the transcription cycle, the mobilization of stored RNAP IIO, and the regulation of transcript elongation and RNA processing is discussed.

Dephosphorylation of RNA polymerase II by CTD-phosphatase FCP1 is inhibited by phospho-CTD associating proteins.

Reversible phosphorylation of the repetitive C-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit plays a key role in the progression of RNAP through the transcription cycle. The level of CTD phosphorylation is determined by multiple CTD kinases and a CTD phosphatase, FCP1. The phosphorylated CTD binds to a variety of proteins including the cis/trans peptidyl-prolyl isomerase (PPIase) Pin1 and enzymes involved in processing of the primary transcript such as the capping enzyme Hce1 and CA150, a nuclear factor implicated in transcription elongation. Results presented here establish that the dephosphorylation of hyperphosphorylated RNAP II (RNAP IIO) by FCP1 is impaired in the presence of Pin1 or Hce1, whereas CA150 has no influence on FCP1 activity. The inhibition of dephosphorylation is observed with free RNAP IIO generated by different CTD kinases as well as with RNAP IIO engaged in an elongation complex. These findings support the idea that specific phospho-CTD associating proteins can differentially modulate the dephosphorylation of RNAP IIO by steric hindrance and may play an important role in the regulation of gene expression.

TFIIF-associating carboxyl-terminal domain phosphatase dephosphorylates phosphoserines 2 and 5 of RNA polymerase II.

The carboxyl-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit undergoes reversible phosphorylation throughout the transcription cycle. The unphosphorylated form of RNAP II is referred to as IIA, whereas the hyperphosphorylated form is known as IIO. Phosphorylation occurs predominantly at serine 2 and serine 5 within the CTD heptapeptide repeat and has functional implications for RNAP II with respect to initiation, elongation, and transcription-coupled RNA processing. In an effort to determine the role of the major CTD phosphatase (FCP1) in regulating events in transcription that appear to be influenced by serine 2 and serine 5 phosphorylation, the specificity of FCP1 was examined. FCP1 is capable of dephosphorylating heterogeneous RNAP IIO populations of HeLa nuclear extracts. The extent of dephosphorylation at specific positions was assessed by immunoreactivity with monoclonal antibodies specific for phosphoserine 2 or phosphoserine 5. As an alternative method to assess FCP1 specificity, RNAP IIO isozymes were prepared in vitro by the phosphorylation of purified calf thymus RNAP IIA with specific CTD kinases and used as substrates for FCP1. FCP1 dephosphorylates serine 2 and serine 5 with comparable efficiency. Accordingly, the specificity of FCP1 is sufficiently broad to dephosphorylate RNAP IIO at any point in the transcription cycle irrespective of the site of serine phosphorylation within the consensus repeat.

The repetitive C-terminal domain of RNA polymerase II: multiple conformational states drive the transcription cycle.

RNA polymerase (RNAP) II is a complex multisubunit enzyme responsible for the synthesis of mRNA in eukaryotic cells. The largest subunit contains at its C-terminus a unique domain, designated the CTD, comprised of tandem repeats of the consensus sequence Tyr(1)Ser(2)Pro(3)Thr(4)Ser(5)Pro(6)Ser(7). This repeat occurs 52 times in mammalian RNAP II. The CTD is subject to extensive phosphorylation at specific points in the transcription cycle by distinct CTD kinases that phosphorylate certain positions within the consensus repeat. The level and pattern of phosphorylation is determined by the concerted action of CTD kinases and CTD phosphatases. The highly dynamic modification by multiple CTD kinases and phosphatases generate distinct conformations of the CTD that facilitate the recruitment of specific macromolecular assemblies to RNAP II. These CTD interacting proteins influence formation of a preinitiation complex at the promoter and couple processing of the primary transcript to the elongation complex.

A novel RNA polymerase II C-terminal domain phosphatase that preferentially dephosphorylates serine 5.

The transcription and processing of pre-mRNA in eukaryotic cells are regulated in part by reversible phosphorylation of the C-terminal domain of the largest RNA polymerase (RNAP) II subunit. The CTD phosphatase, FCP1, catalyzes the dephosphorylation of RNAP II and is thought to play a major role in polymerase recycling. This study describes a family of small CTD phosphatases (SCPs) that preferentially catalyze the dephosphorylation of Ser5 within the consensus repeat. The preferred substrate for SCP1 is RNAP II phosphorylated by TFIIH. Like FCP1, the activity of SCP1 is enhanced by the RAP74 subunit of TFIIF. Expression of SCP1 inhibits activated transcription from a number of promoters, whereas a phosphatase-inactive mutant of SCP1 enhances transcription. Accordingly, SCP1 may play a role in the regulation of gene expression, possibly by controlling the transition from initiation/capping to processive transcript elongation.

Fate of RNA polymerase II stalled at a cisplatin lesion.

Elongating RNA polymerase II blocked by DNA damage in the transcribed DNA strand is thought to initiate the transcription-coupled repair process. The objective of this study is to better understand the sequence of events that occurs during repair from the time RNA polymerase II first encounters the lesion. This study establishes that an immobilized DNA template containing a unique cisplatin lesion can serve as an in vitro substrate for both transcription and DNA repair. RNA polymerase II is quantitatively stalled at the cisplatin lesion during transcription and can be released from the template, along with the nascent transcript, in an ATP-dependent manner. RNA polymerase II stalled at a lesion and containing a dephosphorylated repetitive carboxyl-terminal domain (CTD) appears to be more sensitive toward release. However, a dephosphorylated CTD can become readily phosphorylated in front of the lesion by CTD kinases in the presence of ATP. The observation that RNA polymerase II and transcript release occurs in a TFIIH-deficient repair extract but not in a reconstituted repair system demonstrates that disassembly of the elongation complex can occur independently of the repair process and vice versa. Indeed, the presence of RNA polymerase II at the lesion does not prevent dual incision from occurring. Finally, we also propose that the Cockayne's syndrome B protein factor, believed to be the mammalian transcription repair coupling factor, is neither involved in transcript release nor required for dual incision in the presence of lesionstalled RNA polymerase II in vitro. More likely, it prevents RNA polymerase from backing up when it encounters the lesion. The ability to transcribe and repair the same damaged DNA molecule fixed on beads, along with the fact that the reaction conditions can be freely altered, provides a powerful tool to study the fate of RNA polymerase II blocked on the cisplatin lesion.

Purification and characterization of the human elongator complex.

Human Elongator complex was purified to virtual homogeneity from HeLa cell extracts. The purified factor can exist in two forms: a six-subunit complex, holo-Elongator, which has histone acetyltransferase activity directed against histone H3 and H4, and a three-subunit core form, which does not have histone acetyltransferase activity despite containing the catalytic Elp3 subunit. Elongator is a component of early elongation complexes formed in HeLa nuclear extracts and can interact directly with RNA polymerase II in solution. Several human homologues of the yeast Elongator subunits were identified as subunits of the human Elongator complex, including StIP1 (STAT-interacting protein 1) and IKAP (IKK complex-associated protein). Mutations in IKAP can result in the severe human disorder familial dysautonomia, raising the possibility that this disease might be due to compromised Elongator function and therefore could be a transcription disorder.