Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have isolated the murine cDNA homologue of the human protein tyrosine phosphatase PTP-PEST (MPTP-PEST) from an 18.5-day mouse embryonic kidney library. The cDNA isolated has a single open reading frame predicting a protein of 775 amino acids. When expressed in vitro as a glutathione S-transferase fusion protein, the catalytic domain (residues 1-453) shows intrinsic phosphatase activity. Reverse transcriptase PCR and Northern-blot analysis show that MPTP-PEST mRNA is expressed throughout murine development. Indirect immunofluorescence in COS-1 cells against a heterologous epitope tag attached to the N-terminus of MPTP-PEST, together with cellular fractionation and Western-blot experiments from different murine cell lines, indicate that MPTP-PEST is a free cytosolic protein of 112 kDa. Finally, sequence analysis indicates that the C-terminal portion of the protein contains four regions rich in proline, glutamate, serine and threonine, otherwise known as PEST sequences. These are characteristic of proteins that display very short intracellular half-lives. Despite the presence of these motifs, pulse-chase labelling experiments demonstrate that MPTP-PEST has a half-life of more than 4 h.
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PMID:Murine protein tyrosine phosphatase-PEST, a stable cytosolic protein tyrosine phosphatase. 777 23

Each cycle of transcription appears to be associated with the reversible phosphorylation of the repetitive COOH-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit. The dephosphorylation of RNAP II by CTD phosphatase, therefore, plays an important role in the transcription cycle. The following studies characterize the activity of HeLa cell CTD phosphatase with a special emphasis on the regulation of CTD phosphatase activity. Results presented here suggest that RNAP II contains a docking site for CTD phosphatase that is essential in the dephosphorylation reaction and is distinct from the CTD. This is supported by the observations that (a) phosphorylated recombinant CTD is not a substrate for CTD phosphatase, (b) RNAP IIB, which lacks the CTD, and RNAP IIA are competitive inhibitors of CTD phosphatase and (c) CTD phosphatase can form a stable complex with RNAP II. To test the possibility that the general transcription factors may be involved in the regulation of CTD phosphatase, CTD phosphatase activity was examined in the presence of recombinant or highly purified general transcription factors. TFIIF stimulates CTD phosphatase activity 5-fold. The RAP74 subunit of TFIIF alone contained the stimulatory activity and the minimal region sufficient for stimulation corresponds to COOH-terminal residues 358-517. TFIIB inhibits the stimulatory activity of TFIIF but has no effect on CTD phosphatase activity in the absence of TFIIF. The potential importance of the docking site on RNAP II and the effect of TFIIF and TFIIB in regulating the dephosphorylation of RNAP II at specific times in the transcription cycle are discussed.
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PMID:The activity of COOH-terminal domain phosphatase is regulated by a docking site on RNA polymerase II and by the general transcription factors IIF and IIB. 779 76

The repetitive C-terminal domain (CTD) of RNA polymerase (RNAP) II is extensively phosphorylated concomitant with the initiation of transcription and must be dephosphorylated before RNAP II can begin another round of transcription. A CTD phosphatase was purified more than 7,500-fold from a HeLa cell extract. SDS-polyacrylamide gel electrophoresis shows a predominant protein of 205 kDa and a less abundant protein of 150 kDa co-eluting with the CTD phosphatase activity. Sedimentation and gel filtration analysis suggest that CTD phosphatase has an elongated structure with a M(r) of 200,000. This enzyme is a type 2C phosphatase in that it requires Mg2+ for activity and is resistant to okadaic acid. CTD phosphatase appears to processively dephosphorylate the CTD and is specific in that it does not dephosphorylate phosphorylase a, the alpha or beta subunits of phosphorylase kinase or RNAP II phosphorylated with casein kinase II. CTD phosphatase dephosphorylates RNAP IIO purified from calf thymus or generated in vitro by two previously described CTD kinases. These results suggest that CTD phosphatase has the properties expected for a protein phosphatase that catalyzes the conversion of RNAP IIO to RNAP IIA and may play a key role in the transcription cycle of RNAP II.
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PMID:Purification and characterization of a phosphatase from HeLa cells which dephosphorylates the C-terminal domain of RNA polymerase II. 792 41

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.
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PMID:Enhanced phosphorylation of the C-terminal domain of RNA polymerase II upon serum stimulation of quiescent cells: possible involvement of MAP kinases. 795 47

The transcription initiation factor, TFIIF, is essential not only for the initiation of transcription but also for efficient elongation of mRNA synthesis by mammalian RNA polymerase II and is extensively phosphorylated in vivo. The possible regulation of TFIIF activity by protein phosphorylation was investigated by comparing the biochemical properties of alkaline phosphatase-treated HeLa TFIIF with those of native or bacterially expressed factor. Alkaline phosphatase treatment decreased the size of the large subunit (RAP74) of TFIIF to that of the recombinant protein but did not change the size of the small subunit (RAP30). Both the transcription initiation and elongation stimulating activities of the alkaline phosphatase-treated TFIIF decreased to 15-20% of the native form under conditions in which the amount of TFIIF was rate-limiting for transcription. Furthermore, phosphatase-treated TFIIF assembled the DBPolF complex and bound to RNA polymerase II less efficiently than the native protein. When hybrid TFIIFs were reconstituted using native or recombinant subunits, a native form of RAP74 stimulated both transcription and DBPolF complex formation activity regardless of whether native or recombinant RAP30 was used. We propose that TFIIF activity is regulated by protein phosphorylation, particularly of the RAP74 subunit. The functional role of RAP74 in assembling the preinitiation complex and modulating TFIIF activity is discussed.
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PMID:Regulation of the human general transcription initiation factor TFIIF by phosphorylation. 796 96

Human diploid fibroblasts undergo a limited number of population doublings in vitro and are used widely as a model of cellular aging. Despite growing evidence that cellular aging occurs as a consequence of altered gene expression, little is known about the activity of transcription factors in aging cells. Here, we report a dramatic reduction in the ability of proteins extracted from the nuclei of near-senescent fibroblasts to bind the serum response element which is necessary for serum-induced transcription of the c-fos gene. In contrast, the activities of proteins binding to the RNA polymerase core element, TATA, as well as to the cyclic AMP response element were maintained during cellular aging. While no major differences in the expression of the serum response factor (SRF) that binds the serum response element were seen between early-passage and late-passage cells, hyperphosphorylation of SRF was observed in near-senescent cells. Furthermore, removal of phosphatase inhibitors during the isolation of endogenous nuclear proteins restored the ability of SRF isolated from old cells to bind the SRE. These data, therefore, indicate that hyperphosphorylation of SRF plays a role in altering the ability of this protein to bind to DNA and regulate gene expression in senescent cells.
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PMID:Loss of serum response element-binding activity and hyperphosphorylation of serum response factor during cellular aging. 800 92

Formation of an RNA polymerase II transcription initiation complex requires binding of a polymerase that contains a non-phosphorylated largest subunit carboxyl-terminal domain (CTD). Polymerase binding is followed by elongation after phosphorylation of the CTD by a CTD kinase. Phosphorylation sites are within the repeating heptapeptide motifs which characterize the CTD of all eukaryotic RNA polymerase IIs. In contrast to all other eukaryotes studied, the trypanosome genome contains two genetic loci which encode the large subunit of RNA polymerase II; both genes lack CTD heptapeptide repeat structures. We have examined whether Trypanosoma brucei RNA polymerase II, despite its unique CTD domain, is phosphorylated when isolated from elongating transcription complexes. Elongating trypanosome RNA polymerases were photoaffinity labeled during nuclear run-on assays. The identity of the labeled proteins was established by immunoblotting and immunoprecipitation using polymerase-specific antisera. Analysis of the largest subunit of RNA polymerase II revealed the expected 195-kDa species and an additional larger 220-kDa species. The apparent molecular weight of this larger form of RNA polymerase II decreased incrementally as a function of incubation with increasing concentrations of calf intestinal phosphatase. These results show that extensive phosphorylation of the largest subunit of RNA polymerase-II is a conserved feature between trypanosomes and higher eukaryotes despite the absence of a typical CTD domain.
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PMID:Trypanosoma brucei RNA polymerase II is phosphorylated in the absence of carboxyl-terminal domain heptapeptide repeats. 810 43

Protein kinase(s) and protein phosphatase(s) present in a Xenopus S-100 transcription extract strongly influence promoter-dependent transcription by RNA polymerase I. The protein kinase inhibitor 6-dimethyl-aminopurine causes transcription to increase, while the protein phosphatase inhibitor okadaic acid causes transcription to decrease. Repression is also observed with inhibitor 2, and the addition of extra protein phosphatase 1 stimulates transcription, indicating that the endogenous phosphatase is a type 1 enzyme. Partial fractionation of the system, single-round transcription reactions, and kinetic experiments show that two different steps during ribosomal gene transcription are sensitive to protein phosphorylation: okadaic acid affects a step before or during transcription initiation, while 6-dimethylaminopurine stimulates a process "late" in the reaction, possibly reinitiation. The present results are a clear demonstration that transcription by RNA polymerase I can be regulated by protein phosphorylation.
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PMID:Identification of two steps during Xenopus ribosomal gene transcription that are sensitive to protein phosphorylation. 811 32

The phosphorylation of the C-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II has been investigated in HeLa cells exposed to heat shock. In control cells, the phosphorylated subunit, IIo, and the dephosphorylated subunit, IIa, were found in similar amounts. During heat shock, however, the phosphorylated subunit, IIo, accumulated, whereas the amount of IIa subunit decreased. Since phosphorylation of the CTD had been suggested to play a role in the initiation of transcription and since heat shock was known to perturb gene expression at the level of transcription, the phosphorylation state of RNA polymerase II was examined in cells that had been treated with various inhibitors of transcription. Under normal growth temperature, actinomycin D (over 0.1 microgram/ml) and okadaic acid, a phosphatase inhibitor, were found to inhibit polymerase dephosphorylation. Whereas 5,6-dichlorobenzimidazole riboside (DRB), N-(2-[Methylamino]ethyl)-5-isoquinolinesulfonamide (H-8), and actinomycin D (over 5 micrograms/ml) were found to inhibit polymerase phosphorylation. Actinomycin D concentrations, which inhibited the dephosphorylation process, were lower than those required to inhibit the phosphorylation process. In contrast, during heat shock or exposure to sodium arsenite, a chemical inducer of the heat-shock response, the phosphorylated subunit, IIo, accumulated even in the presence of inhibitors of transcription such as DRB, H-8, and actinomycin D. These experiments demonstrated the existence of a heat-shock-induced CTD-phosphorylation process that might contribute to the regulation of transcription during stress.
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PMID:Phosphorylation of the RNA polymerase II largest subunit during heat shock and inhibition of transcription in HeLa cells. 812 66

A new cloning strategy is described which utilizes direct selection of recombinants for shotgun sequencing in the filamentous bacteriophage M13. Direct selection is accomplished by insertional inactivation of the M13 gene X protein, a powerful inhibitor of phage-specific DNA synthesis when overproduced. An extra copy of gene X was inserted into the intergenic region of M13 and placed under the control of the bacteriophage T7 gene 10 promoter and RBS. Random fragments are cloned into the EcoRV cloning site of the new gene X cistron and recombinants are selected in an E. coli male strain producing T7 RNA polymerase. Cloning efficiencies obtained with M13-100 or phosphatase-treated M13mp19 vector are comparable. The direct selection capability of M13-100 was demonstrated to have the following advantages: (a) consistently achieved high ratios of recombinants to religated vector in the libraries, averaging about 500:1 (0.2% background), and (b) the elimination of the need for phosphatase treatment of the vector to reduce background. The direct selection strategy significantly improves the efficiency of shotgun library construction in M13, and should therefore facilitate the cloning aspects of large scale sequencing projects.
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PMID:A direct selection strategy for shotgun cloning and sequencing in the bacteriophage M13. 812 45


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