Michael E. Dahmus

Reversible Phosphorylation of the C-terminal Domain of RNA Polymerase II

RNA polymerase (RNAP) II is responsible for the synthesis of pre-mRNA in eukaryotic cells. The subunit structure of RNAP II is similar to that of other RNAPs in that it is comprised of two large subunits with a molecular weight in excess of 100,000 and a collection of smaller subunits (1, 2). However, the largest subunit of RNAP II is unique in that it contains an unusual domain at its C terminus comprised of tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser (3). The consensus repeat has been conserved in evolution although the number of repeats present varies in different species. RNA polymerase II of mammalian cells contains 52 copies of the consensus repeat, and yeast contains 26–27 copies, whereas other eukaryotes contain an intermediate number of repeats. Although this C-terminal domain (CTD) plays an essential role in transcription catalyzed by RNAP II, it is absent from RNAPs I and III. The CTD of yeast and mammalian RNAP II was first reported about 10 years ago and is shown in Fig. 1 (4, 5). This domain has provided a focal point for the analysis of RNAP II structure-function relationships. Although our understanding of the CTD has increased considerably in the ensuing 10 years, its precise role in transcription remains to be established.

Rapid visualization of protein bands in preparative SDS-polyacrylamide gels

Abstract A rapid sensitive method has been developed for the detection of protein bands in sodiumdodecyl sulfate (SDS)-polyacrylamide gels that is suitable for the recovery of individual polypeptides on a preparative scale. The process is reversible and allows for the complete recovery of unmodified protein from preparative gels. The visualization is rapid, requiring between 30 and 40 min, and sensitive, detecting as little protein as 0.1 μ g/mm 3 . The procedure employs high concentrations of sodium acetate.

Phosphorylation of the C-terminal domain of RNA polymerase II

The CTD has become a focal point in the analysis of RNAP II. The unusual properties of the CTD, including its unique structure and high level of phosphorylation, have stimulated interest in understanding the role this domain plays in the transcription of protein-coding genes. Research during the past ten years suggests that the CTD may function at multiple steps in the transcription cycle and that its involvement is promoter dependent. The general idea, for which there is now considerable support, is that the CTD mediates the interaction of RNAP II with the transcription apparatus and that these interactions are influenced by the phosphorylation that occurs throughout the CTD. The temporal relationship between phosphorylation of the CTD and the progression of RNAP II through the transcription cycle has been established in a general sense. However, it is not clear that the modifications that occur at a given time are causally related to the progression of RNAP II beyond that point in the transcription cycle. The idea that phosphorylation of the CTD mediates the release of RNAP II from the preinitiation complex is an attractive one and consistent with a number of experimental results. However, an increasing number of observations suggest that CTD phosphorylation and promoter clearance may not be causally related. One possibility is that even though phosphorylation occurs concomitant with transcript initiation it plays no real role in the initiation process and is necessary only to establish an elongation competent form of the enzyme. Alternatively, CTD phosphorylation may play an essential role in the release of RNAP II from preinitiation complexes in vivo but may be dispensable in defined in vitro transcription systems. Finally it may be important to distinguish between promoter clearance as defined by RNAP moving off the transcriptional start site and the complete disruption of interactions between RNAP II and the preinitiation complex. Because of the extended nature of the CTD, RNAP II may remain tethered to factors assembled on the promoter even though a short transcript has been synthesized. Clearly additional research is necessary to (a) define the contacts made by the CTD in preinitiation complexes, (b) understand the relationship between the disruption of these contacts and CTD phosphorylation and (c) understand biochemically what is required to generate an elongation competent form of RNAP II. The possibility that the CTD plays a role in transcript elongation has been proposed since the discovery of the CTD [15].(ABSTRACT TRUNCATED AT 400 WORDS)

The Role of Multisite Phosphorylation in the Regulation of RNA Polymerase II Activity

Publisher Summary This chapter discusses (a) the relationship between the state of carboxy-terminal domain (CTD) phosphorylation and the progression of RNAP II through the transcription cycle; (b) the enzymes involved in modulating the state of CTD phosphorylation; and (c) the potential consequences of modifications that occur within the CTD. RNA polymerase (RNAP) II is structurally distinct from both RNAP I that transcribes heavy ribosomal RNA (rRNA) and RNAP III that transcribes a variety of small RNAs, including 5-S rRNA and transfer RNA (tRNA). RNAP II transcribes the greatest diversity of genes, and hence must be able to assemble into preinitiation complexes on a variety of different promoters. The largest subunit of RNAP I1 contains at its c-terminus an unusual domain, consisting of multiple heptapeptide repeats of the consensus sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This c-terminal domain (CTD) is conserved in evolution and is essential for cell viability. The functions for the CTD is regulated by reversible phosphorylation that include: (a) mediating the interaction of RNAP II, with the preinitiation complex; (b) mediating the release of RNAP I1 from the initiated complex; (c) facilitating the progression of RNAP II through nucleosomes (X); (d) affecting cotranscriptional splicing, by facilitating the association of splicing factors, with the elongation complex; and (e) influencing the specificity of pausing and termination.

Enhanced phosphorylation of the C-terminal domain of RNA polymerase II upon serum stimulation of quiescent cells: possible involvement of MAP kinases.

The largest subunit of RNA polymerase (RNAP) II contains at it C‐terminus an unusual domain comprising tandem repeats of the consensus sequence Tyr‐Ser‐Pro‐Thr‐Ser‐Pro‐Ser. This C‐terminal domain (CTD) can undergo phosphorylation at multiple sites giving rise to a form of the enzyme designated RNAP IIO. The unphosphorylated form is designated RNAP IIA. The largest subunits of RNAPs IIO and IIA are designated IIo and IIa, respectively. In quiescent NIH 3T3 fibroblasts, subunits IIo and IIa are present in comparable amounts. Upon serum stimulation, the amount of subunit IIo increases markedly and remains elevated for several hours. The increase of subunit IIo also occurs in transcription‐inhibited cells and, therefore, is not a consequence of serum‐activated transcription. This observation suggests that serum stimulation activates a CTD kinase and/or inhibits a CTD phosphatase. This hypothesis is supported by the finding that serum stimulates phosphorylation of a beta‐galactosidase‐CTD fusion protein expressed in these cells. Furthermore, an enhanced CTD kinase activity was discovered in lysates from serum‐stimulated fibroblasts and was found to copurify with MAP kinases on a Mono Q column and to bind to anti‐MAP kinase antibodies. The idea that MAP kinases phosphorylate the CTD in vivo is supported by the observation that subunit IIa, but not subunit IIb which lacks the CTD, is phosphorylated at multiple sites by purified MAP kinase. Consequently, the MAP kinases are a new class of CTD kinases which appear to be involved in the phosphorylation of RNAP II following serum stimulation. This phosphorylation may contribute to the transcriptional activation of serum‐stimulated genes.

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 Cockaynes 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.

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.

Regulation of Carboxyl-terminal Domain Phosphatase by HIV-1 Tat Protein

The phosphorylation state of the carboxyl-terminal domain (CTD) of RNA polymerase (RNAP) II is directly linked to the phase of transcription being carried out by the polymerase. Enzymes that affect CTD phosphorylation can thus play a major role in the regulation of transcription. A previously characterized HeLa CTD phosphatase has been shown to processively dephosphorylate RNAP II and to be stimulated by the 74-kDa subunit of TFIIF. This phosphatase is shown to be comprised of a single 150-kDa subunit by the reconstitution of catalytic activity from a SDS-polyacrylamide gel electrophoresis purified protein. This subunit has been previously cloned and shown to interact with the HIV Tat protein. To determine whether this interaction has functional consequences, the effect of Tat on CTD phosphatase was investigated. Full-length Tat-1 protein (Tat 86R) strongly inhibits the activity of CTD phosphatase. Point mutations in the activation domain of Tat 86R, which reduce the ability of Tat to transactivate in vivo, diminish its ability to inhibit CTD phosphatase. Furthermore, a deletion mutant missing most of the activation domain is unable to inhibit CTD phosphatase activity. The ability of Tat to transactivatein vitro also correlates with the strength of inhibition of CTD phosphatase. These results are consistent with the hypothesis that Tat-dependent suppression of CTD phosphatase is part of the transactivation function of Tat.

Synthesis and characterization of a DNA probe complementary to rat liver 28S ribosomal RNA

Abstract Conditions for the production of a complementary DNA sequence for use in studies of ribosomal RNA are described. E . coli DNA polymerase I is used to transcribe highly purified 28S ribosomal RNA from rat liver. The reaction is sensitive to the tertiary structure of the rRNA template-primer. The complementary DNA hybridizes to its rRNA template with a R o t 1 2 of 0.02. The hybrid formed between 28S ribosomal RNA and complementary DNA has a Tm of 73°C. The probe reacts with total rat nuclear RNA with a R o t 1 2 of 1.0.

Transcription-Independent RNA Polymerase II Dephosphorylation by the FCP1 Carboxy-Terminal Domain Phosphatase in Xenopus laevis Early Embryos

ABSTRACT The phosphorylation of the RNA polymerase II (RNAP II) carboxy-terminal domain (CTD) plays a key role in mRNA metabolism. The relative ratio of hyperphosphorylated RNAP II to hypophosphorylated RNAP II is determined by a dynamic equilibrium between CTD kinases and CTD phosphatase(s). The CTD is heavily phosphorylated in meioticXenopus laevis oocytes. In this report we show that the CTD undergoes fast and massive dephosphorylation upon fertilization. A cDNA was cloned and shown to code for a full-length xFCP1, theXenopus orthologue of the FCP1 CTD phosphatases in humans and Saccharomyces cerevisiae. Two critical residues in the catalytic site were identified. CTD phosphatase activity was observed in extracts prepared from Xenopuseggs and cells and was shown to be entirely attributable to xFCP1. The CTD dephosphorylation triggered by fertilization was reproduced upon calcium activation of cytostatic factor-arrested egg extracts. Using immunodepleted extracts, we showed that this dephosphorylation is due to xFCP1. Although transcription does not occur at this stage, phosphorylation appears as a highly dynamic process involving the antagonist action of Xp42 mitogen-activated protein kinase and FCP1 phosphatase. This is the first report that free RNAP II is a substrate for FCP1 in vivo, independent from a transcription cycle.