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Results for “RNA Polymerase II”

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PubMed1989

Factors involved in specific transcription by mammalian RNA polymerase II. Factors IIE and IIF independently interact with RNA polymerase II.

The purification and characterization of transcription factor IIF (TFIIF), a factor required for transcription by the RNA polymerase II machinery, is described. TFIIF was isolated from the previously described IIE protein fraction. TFIIF enters into the transcription cycle via a preinitiation complex, and it is required for the formation of a complex capable of initiating transcription in the presence of heparin concentrations that inhibit the action of a free factor. TFIIF and TFIIE independently interacted with purified RNA polymerase II. TFIIF and TFIIE were both required for transcription of several class II promoters, including a promoter that lacks the conserved TATA box. Interestingly TFIIF was absolutely required for the formation of a preinitiation complex; however, it also affected the elongation phase of the transcription cycle. TFIIF, together with the previously described elongation factor TFIIS, was required for efficient elongation.

Adenoviruses, Human↗

PubMed1998

Yeast transcript elongation factor (TFIIS), structure and function. II: RNA polymerase binding, transcript cleavage, and read-through.

The transcriptionally active fragment of the yeast RNA polymerase II transcription elongation factor, TFIIS, comprises a three-helix bundle and a zinc ribbon motif joined by a linker region. We have probed the function of this fragment of TFIIS using structure-guided mutagenesis. The helix bundle domain binds RNA polymerase II with the same affinity as does the full-length TFIIS, and this interaction is mediated by a basic patch on the outer face of the third helix. TFIIS mutants that were unable to bind RNA polymerase II were inactive for transcription activity, confirming the central role of polymerase binding in the TFIIS mechanism of action. The linker and zinc ribbon regions play roles in promoting cleavage of the nascent transcript and read-through past the block to elongation. Mutation of three aromatic residues in the zinc ribbon domain (Phe269, Phe296, and Phe308) impaired both transcript cleavage and read-through. Mutations introduced in the linker region between residues 240 and 245 and between 250 and 255 also severely impaired both transcript cleavage and read-through activities. Our analysis suggests that the linker region of TFIIS probably adopts a critical structure in the context of the elongation complex.

Models, Molecular↗

PubMed1980

Transcription of simian virus 40 DNA by wheat germ RNA polymerase II. Priming of RNA synthesis by the 3'-hydroxyl of DNA at single strand nicks.

Linear simian virus 40 DNA has been transcribed in vitro with wheat germ RNA polymerase II. Transcription products have been fractionated on polyacrylamide gels and several discrete sized RNA bands are seen. The RNA band pattern is affected dramatically by deoxyribonuclease treatment during RNA isolation. This is because most of the RNA synthesized is covalently linked to DNA. This linkage has been demonstrated by density analysis in formaldehyde-CsCl gradients and by incorporation of alkali-stable ribonucleotides into DNA. The linear DNA templates transcribed were generated by treatment of circular DNA with restriction enzymes which, in addition to cutting once at a single primary site, were found also to produce single strand nicks at specific secondary sites. The discrete sized RNA bands observed result from initiation at these nicks and terminated at DNA ends. There are two modes of nick-dependent initiation. In one mode the 3'-hydroxyl terminus of the DNA at a single strand nick serves as a primer for the extension of an RNA chain. In a second mode de novo initiation of an RNA chain is promoted at the nick. RNAs which are not primed initiate predominantly with GTP. The catalytic action of wheat germ RNA polymerase II is similar to that of Escherichia coli core RNA polymerase which has also been shown to synthesize primarily RNA which is covalently linked to DNA.

Base Sequence↗

PubMed1975

A new method for the large-scale purification of wheat germ DNA-dependent RNA polymerase II.

An improved method for the purification of the alpha-amanitin-sensitive deoxyribonucleic acid dependent ribonucleic acid polymerase [ribonucleosidetriphosphate:RNA-nucleotidyltransferase, EC 2.7.7.6-A1 (RNA polymerase II or RNA polymerase B) from wheat germ is presented. The method involves homogenization of wheat germ in a buffer of moderate ionic strength, precipitation of RNA polymerase with Polymin P (a polyethylenimine), elution of RNA polymerase from the Polymin P precipitate, ammonium sulfate precipitation, and chromatography on DEAE-cellulose and phosphocellulose. RNA polymerase II is purified over 4000-fold with a 60% recovery, resulting in a yield of 25-30 mg of RNA polymerase from 1 kg of starting material.

Chromatography, DEAE-Cellulose↗

PubMed2002

Yhh1p/Cft1p directly links poly(A) site recognition and RNA polymerase II transcription termination.

RNA polymerase II (pol II) transcription termination requires co-transcriptional recognition of a functional polyadenylation signal, but the molecular mechanisms that transduce this signal to pol II remain unclear. We show that Yhh1p/Cft1p, the yeast homologue of the mammalian AAUAAA interacting protein CPSF 160, is an RNA-binding protein and provide evidence that it participates in poly(A) site recognition. Interestingly, RNA binding is mediated by a central domain composed of predicted beta-propeller-forming repeats, which occurs in proteins of diverse cellular functions. We also found that Yhh1p/Cft1p bound specifically to the phosphorylated C-terminal domain (CTD) of pol II in vitro and in a two-hybrid test in vivo. Furthermore, transcriptional run-on analysis demonstrated that yhh1 mutants were defective in transcription termination, suggesting that Yhh1p/Cft1p functions in the coupling of transcription and 3'-end formation. We propose that direct interactions of Yhh1p/Cft1p with both the RNA transcript and the CTD are required to communicate poly(A) site recognition to elongating pol II to initiate transcription termination.

Actins↗

PubMed1981

A method for assaying DNA-dependent RNA polymerase II in crude extracts of Drosophila melanogaster adults: its use in identifying mutants with an altered RNA polymerase II.

A method for assaying Drosophila melanogaster adult DNa-dependent RNA polymerase II in crude extracts from as a few as two females or three males is described. Preparation of the extracts involves incubating homogenates at 25 C for 60 min and subsequent treatment with Macaloid. Eighty-five percent of the activity in the extracts is inhibited by 1 microgram/ml alpha-amanitin and this fraction is attributed to RNA polymerase II. RNA polymerase II activity in the extracts shows a good dose dependence and a partial dependence on added DNA, Mn2+, and all four ribonucleoside triphosphates. The kinetics of heat inactivation of RNA polymerase II in crude extracts could be reproducibly measured. Flies of different genotypes had different initial rates of RNA polymerase II heat inactivation. The isolation of Drosophila melanogaster alpha-amanitin-resistant mutants is also reported. Using the assay described in this paper, it appears that the basis for the resistance is an altered RNA polymerase II. The mutation has been mapped to the third chromosome by chromosome replacement.

Amanitins↗

PubMed1995

A novel activity associated with RNA polymerase II elongation factor SIII. SIII directs promoter-independent transcription initiation by RNA polymerase II in the absence of initiation factors.

General transcription factor SIII, a heterotrimer of 110-, 18-, and 15-kDa subunits, was shown previously to stimulate the overall rate of RNA chain elongation by RNA polymerase II by suppressing transient pausing by polymerase at many sites along DNA templates (Bradsher, J. N., Jackson, K. W., Conaway, R. C., and Conaway, J. W. (1993) J. Biol. Chem. 268, 25587-25593). In this report, SIII is shown to possesses the novel ability to direct robust but promiscuous transcription by RNA polymerase II on duplex DNA templates in the absence of initiation factors. Mechanistic studies reveal that SIII promotes RNA synthesis by substantially increasing the efficiency with which RNA polymerase II initiates promoter-independent transcription from the ends of duplex DNA. Remarkably, SIII appears to have a negligible effect on de novo synthesis of end-to-end transcripts. Instead, analysis of reaction products indicates that SIII is capable of promoting a dramatic increase in the ability of RNA polymerase II to extend the 3'-hydroxyl termini of duplex DNA fragments, in a template-directed reaction exhibiting no strong preference for 3'-protruding, 3'-recessed, or blunt DNA ends. Although RNA polymerase II has been shown previously to catalyze primer-dependent transcription, SIII is the first eukaryotic transcription factor found to promote this reaction. Based on these findings, we propose that SIII may suppress transient pausing by RNA polymerase II by helping to maintain the 3'-hydroxyl terminus of the nascent RNA chain in its proper position in the polymerase active site.

Aging↗

PubMed1993

RNA polymerase II transcription cycles.

RNA polymerase II interacts with transcription factors to initiate the synthesis of mRNA. These interactions are cyclic, involving multiple RNA polymerase subunits and general transcription factors. Phosphorylation of the RNA polymerase II carboxyl-terminal domain may regulate these interactions.

Amino Acid Sequence↗

PubMed1984

Enhancement of the frequency of initiation by a stimulatory protein of RNA polymerase II.

By use of purified RNA polymerase II, it was demonstrated that S-II, a stimulatory protein of RNA polymerase II, enhanced the frequency of initiation of transcription from discrete sites on the promoter region of the silk fibroin gene integrated in a supercoiled plasmid DNA in the presence of manganese. In the absence of S-II, RNA polymerase II preferentially initiated RNA synthesis from site +25, 25 bases downstream from the cap site. Of these initiation sites, the initiation from site +25 was not affected by S-II, suggesting that site +25 is structurally different from other initiation sites, including the cap site, and that S-II modifies the latter sites to the same structure as that of site +25. Direct interaction between S-II and DNA in the initiation complex was shown by demonstrating a conformational change of S-II on its interaction with DNA; namely, S-II in the initiation complex was as sensitive to chymotryptic digestion as S-II interacting with DNA, whereas free S-II was completely insensitive to chymotryptic digestion.

Animals↗

PubMed1995

News on initiation and elongation of transcription by RNA polymerase II.

Transcription by RNA polymerase II is a complex process that requires additional factors to initiate transcription at the promoters. New developments in the past year have furthered our understanding of the functions of the transcription factors and provided more insights into the mechanisms involved in the regulation of initiation and elongation of transcription. One of the most significant advances of the past year was the discovery of the involvement of the general transcription factor TFIIH in DNA excision repair. Surprisingly, studies aimed at identifying the kinase activity within TFIIH responsible for phosphorylating the carboxy-terminal domain of RNA polymerase II revealed it to be the MO15/Cdk7 kinase and its partner, cyclin H. These exciting observations suggest a paradigm for linking transcription, DNA excision repair and cell cycle progression through one pivotal factor.

Humans↗

PubMed1997

Glutamic acid-371 of the barnase homology domain in RNA polymerase II is not required for SII-activated RNA cleavage.

RNA polymerase II contains a ribonuclease activity which is stimulated by the transcription elongation factor SII. This nuclease shortens the nascent RNA and facilitates relief of transcriptional arrest by allowing the enzyme to make multiple attempts to read through an obstacle to transcription. The catalytic center of this ribonuclease is unknown, although a region of the enzyme's second largest subunit shares local sequence similarly with barnase and other bacterial ribonucleases. To test the role of the barnase homology region in SII-activated cleavage, we engineered a single amino acid change in the Saccharomyces cerevisiae enzyme at a position homologous to a catalytic residue of barnase (Glu-371) and has been suggested as a participant in active site chemistry of RNA polymerase II. We purified RNA polymerase II from mutant yeast and assayed its ability to cleave and re-extend the nascent RNA following SII treatment. We find no defects in this function of the mutant enzyme, suggesting that the barnase homology region does not represent the active site of the SII-activated nuclease. These mutant yeast cells were also resistant to mycophenolic acid, which slows the growth of some yeast mutants bearing elongation defective RNA polymerase II or mutant elongation factor SII.

Amino Acid Sequence↗

PubMed2005

The carboxyl-terminal domain of RNA polymerase II is phosphorylated by a complex containing cdk9 and infected-cell protein 22 of herpes simplex virus 1.

The infected-cell protein 22 (ICP22), a regulatory protein encoded by the alpha22 gene of herpes simplex virus 1, is required for the optimal expression of a set of late viral proteins that includes the products of the U(S)11, U(L)38, and U(L)41 genes. ICP22 has two activities. Thus, ICP22 and the U(L)13 protein kinase mediate the activation of cdc2 and degradation of its partners, cyclins A and B. cdc2 and its new partner, the DNA polymerase accessory factor (U(L)42), bind topoisomerase IIalpha in an ICP22-dependent manner. In addition, ICP22 and U(L)13 mediate an intermediate phosphorylation of the carboxyl terminus of RNA polymerase II (RNA POL II). Here we report another function of ICP22. Thus, ICP22 physically interacts with cdk9, a constitutively active cyclin-dependent kinase involved in transcriptional regulation. A protein complex containing ICP22 and cdk9 phosphorylates in vitro the carboxyl-terminal domain of RNA POL II in a viral U(S)3 protein kinase-dependent fashion. Finally, the carboxyl-terminal domain of RNA POL II fused to glutathione S-transferase is phosphorylated in reaction mixtures containing complexes pulled down with ICP22 or cdk9 immune precipitated from lysates of wild-type parent virus or deltaU(L)13 but not deltaU(S)3 mutant-infected cells. The experiments described here place ICP22 and cdk9 in a complex with the carboxyl-terminal domain of RNA POL II. At the same time we confirm the requirement of ICP22 and the U(L)13 protein kinase in the posttranslational modification of RNA POL II that alters its electrophoretic mobility, although U(S)3 kinase appears to play a role in a cell-type-dependent fashion.

Cell Line↗

PubMed2005

RNA polymerase II synthesizes antisense RNA in Plasmodium falciparum.

The recent identification of antisense RNA in the transcriptomes of many eukaryotes has generated enormous interest. The presence of antisense RNA in Plasmodium falciparum, the causative agent of severe malaria, remains controversial. Elucidation of the mechanism of antisense RNA in P. falciparum synthesis is critical in order to demonstrate the origin and function of these transcripts. Therefore, a systematic analysis of antisense and sense RNA synthesis was performed using direct labeling experiments. Nuclear run on experiments with single-stranded DNA probes demonstrated that antisense RNA is synthesized in the nucleus at several genomic loci. Antisense RNA synthesis is sensitive to the potent RNA polymerase II inhibitor alpha-amanitin. Antisense and sense transcription was also detected in nuclei isolated from synchronized parasites, suggesting concurrent synthesis. In summary, our experiments directly demonstrate that antisense RNA synthesis is a common transcriptional phenomenon in P. falciparum, and is catalyzed by RNA polymerase II.

Animals↗

PubMed1989

An enlarged largest subunit of Plasmodium falciparum RNA polymerase II defines conserved and variable RNA polymerase domains.

We have isolated the gene encoding the largest subunit of RNA polymerase II from Plasmodium falciparum. The RPII gene is expressed in the asexual erythrocytic stages of the parasite as a 9 kb mRNA, and is present as a single copy gene located on chromosome 3. The P. falciparum RPII subunit is the largest (2452 amino acids) eukaryotic RPII subunit, and it contains enlarged variable regions that clearly separate and define five conserved regions of the eukaryotic RPII largest subunits. A distinctive carboxyl-terminal domain contains a short highly conserved heptapeptide repeat domain which is bounded on its 5' side by a highly diverged heptapeptide repeat domain, and is bounded on its 3' side by a long carboxyl-terminal extension.

Amino Acid Sequence↗

PubMed1998

Factors associated with the mammalian RNA polymerase II holoenzyme.

The RNA polymerase II (Pol II) holoenzyme in yeast is an essential transcriptional regulatory complex which has been defined by genetic and biochemical approaches. The mammalian counterpart to this complex, however, is less well defined. Experiments herein demonstrate that, along with Pol II and SRB proteins, proteins associated with transcriptional regulation as cofactors are associated with the Pol II holoenzyme. Earlier experiments have demonstrated that the breast cancer-associated tumor suppressor BRCA1 and the CREB binding protein (CBP) were associated with the holoenzyme complex. The protein related to CBP, the E1A-associated p300 protein, is shown in these experiments to be associated with the holoenzyme complex as well as the BRG1 subunit of the chromatin remodeling SWI/SNF complex. Importantly, the Pol II holoenzyme complex does not contain some factors previously reported as stoichiometric components of the holoenzyme complex, most notably the proteins which function in repair of damaged DNA, such as PCNA, RFC and RPA. The presence of the p300 coactivator and the chromatin-modifying BRG1 protein support a role for the Pol II holoenzyme as a key target for regulation by enhancer binding proteins.

BRCA1 Protein↗

PubMed1983

Protein kinase NII. Interaction with RNA polymerase II and contribution to immunological cross-reactivity of RNA polymerases I and II.

The interaction between antibodies directed against RNA polymerase I purified from Morris hepatoma 3924A and homologous RNA polymerase II was investigated. The activity of partially purified polymerase II was inhibited by the antibodies. In contrast, the reaction catalyzed by the purified enzyme was not affected. Partially purified polymerase II preparations contained a protein kinase activity. Sucrose gradient centrifugation in the presence of 0.3 M KCl resulted in complete separation of RNA polymerase II from protein kinase as well as in complete loss of sensitivity to the anti-RNA polymerase I antibodies. The protein kinase possessed reaction characteristics similar to those of the NII protein kinase (Rose, K.M., Bell, L.E., Siefken, D.A. and Jacob, S.T. (1981) J. Biol. Chem. 256, 7468-7477) which is associated with hepatoma RNA polymerase I (Rose, K.M., Stetler, D.A. and Jacob, S.T. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2833-2837). The activities of both kinases were inhibited to the same extent by anti-RNA polymerase I antibodies and polypeptides of Mr 42 000 and 25 000, present in both kinase preparations, formed immune complexes with the antisera. Readdition of protein kinase NII to purified polymerase II resulted in phosphorylation of the polymerase and a concomitant enhancement of RNA synthesis. After addition of the kinase, RNA polymerase II activity was again sensitive to anti-RNA polymerase I antibodies. Upon reacting with protein kinase NII, RNA polymerase II polypeptides could be detected in immune complexes with anti-RNA polymerase I antibodies. These data indicate that protein kinase NII is associated with RNA polymerase II during early stages of purification and is at least partially responsible for the immunological cross-reactivity of RNA polymerases I and II.

Animals↗

PubMed1998

Regulatory targets in the RNA polymerase II holoenzyme.

The RNA polymerase II holoenzyme is the form of polymerase recruited to promoters for protein-coding genes. Several targets of mammalian activators, previously called coactivators, turn out to be subunits of the holoenzyme which activators use to recruit and regulate the holoenzyme. Several of these newly identified holoenzyme components have been implicated in human disease.

Animals↗

PubMed2004

Rpb4 and Rpb7: subunits of RNA polymerase II and beyond.

RNA Polymerase II (pol II) is a large multi-subunit complex that is responsible for the synthesis of all eukaryotic mRNAs. Its correct and timely recruitment to promoter regions is a crucial step of transcription regulation, involving complicated and well-controlled networks of protein-DNA and protein-protein interactions. The best-studied pol II is the yeast complex consisting of 12 subunits (Rpb1-12). Rpb4 and Rpb7 form a dissociable heterodimer (Rpb4/7). The unique location of Rpb4/7 within the transcription initiation complex, and its capacity to interact with various transcription factors, suggest that it provides important links to the network of interactions that control transcription initiation. Moreover, Rpb4/7 executes some non-transcriptional activities, including mRNA transport. Hence, Rpb4/7 functions at the interface of transcriptional and post-transcriptional machinery.

Animals↗