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PubMed2006

Core promoter-selective RNA polymerase II transcription.

The initiation of mRNA synthesis in eukaryotic cells is a complex and highly regulated process that requires the assembly of general transcription factors and RNAP II (RNA polymerase II; also abbreviated as Pol II) into a pre-initiation complex at the core promoter. The core promoter is defined as the minimal DNA region that is sufficient to direct low levels of activator-independent (basal) transcription by RNAP II in vitro. The core promoter typically extends approx. 40 bp up- and down-stream of the start site of transcription and can contain several distinct core promoter sequence elements. Core promoters in higher eukaryotes are highly diverse in structure, and each core promoter sequence element is only found in a subset of genes. So far, only TATA box and INR (initiator) element have been shown to be capable of directing accurate RNAP II transcription initiation independent of other core promoter elements. Computational analysis of metazoan genomes suggests that the prevalence of the TATA box has been overestimated in the past and that the majority of human genes are TATA-less. While TATA-mediated transcription initiation has been studied in great detail and is very well understood, very little is known about the factors and mechanisms involved in the function of the INR and other core promoter elements. Here we summarize our current understanding of the factors and mechanisms involved in core promoter-selective transcription and discuss possible pathways through which diversity in core promoter architecture might contribute to combinatorial gene regulation in metazoan cells.

Animals↗
PubMed2000

A regulatory shortcut between the Snf1 protein kinase and RNA polymerase II holoenzyme.

RNA polymerase II holoenzymes respond to activators and repressors that are regulated by signaling pathways. Here we present evidence for a "shortcut" mechanism in which the Snf1 protein kinase of the glucose signaling pathway directly regulates transcription by the yeast holoenzyme. In response to glucose limitation, the Snf1 kinase stimulates transcription by holoenzyme that has been artificially recruited to a reporter by a LexA fusion to a holoenzyme component. We show that Snf1 interacts physically with the Srb/mediator proteins of the holoenzyme in both two-hybrid and coimmunoprecipitation assays. We also show that a catalytically hyperactive Snf1, when bound to a promoter as a LexA fusion protein, activates transcription in a glucose-regulated manner; moreover, this activation depends on the integrity of the Srb/mediator complex. These results suggest that direct regulatory interactions between signal transduction pathways and RNA polymerase II holoenzyme provide a mechanism for transcriptional control in response to important signals.

Bacterial Proteins↗
PubMed1994

TFIIF-TAF-RNA polymerase II connection.

RNA polymerase transcription factor IIF (TFIIF) is required for initiation at most, if not all, polymerase II promoters. We report here the cloning and sequencing of genes for a yeast protein that is the homolog of mammalian TFIIF. This yeast protein, previously designated factor g, contains two subunits, Tfg1 and Tfg2, both of which are required for transcription, essential for yeast cell viability, and whose sequences exhibit significant similarity to those of the mammalian factor. The yeast protein also contains a third subunit, Tfg3, which is less tightly associated and at most stimulatory to transcription, dispensable for cell viability, and has no known counterpart in mammalian TFIIF. Remarkably, the TFG3 gene encodes yeast TAF30, and furthermore, is identical to ANC1, a gene implicated in actin cytoskeletal function in vivo (Welch and Drubin 1994). Tfg3 is also a component of the recently described mediator complex (Kim et al. 1994), whose interaction with the carboxy-terminal repeat domain of RNA polymerase II enables transcriptional activation. Deletion of TFG3 results in diminished transcription in vivo.

Amino Acid Sequence↗
PubMed1993

Identification of a 3'-->5' exonuclease activity associated with human RNA polymerase II.

Human RNA polymerase II is shown to be associated with a 3'-->5' exonuclease activity that removes nucleoside 5'-monophosphates from the 3' end of the transcripts in isolated ternary complexes. This activity is stimulated by SII, a protein that acts as a transcription elongation factor in vitro. In addition, we show that another transcription factor, TFIIF, stimulates a competing pyrophosphorolysis reaction. These findings raise interesting questions about the roles of these activities in vivo, including the possibility that this RNA polymerase may proofread the nascent transcript.

Adenoviridae↗
PubMed2002

Functional interaction of yeast pre-mRNA 3' end processing factors with RNA polymerase II.

The RNA polymerase II CTD is essential for 3' end cleavage of metazoan pre-mRNAs and binds 3' end processing factors in vitro. We show genetic and biochemical interactions between the CTD and the Pcf11 subunit of the yeast cleavage/polyadenylation factor, CFIA. In vitro binding to Pcf11 required phosphorylation of the CTD on Ser2 in the YSPTSPS heptad repeats. Deletion of the yeast CTD reduced the efficiency of cleavage at poly(A) sites, and the length of poly(A) tails suggesting that it helps couple 3' end formation with transcription. Consistent with this model, the 3' end processing factors CFIA, CFIB, and PFI were recruited to genes progressively, starting at the 5' end, in a process that required ongoing transcription.

Animals↗
PubMed1997

Selective targeting and inhibition of yeast RNA polymerase II by RNA aptamers.

To probe the complex nucleic acid binding domains of yeast RNA polymerase II (Pol II), we have isolated in the presence of heparin RNA molecules that selectively bind to yeast Pol II. A class of RNA molecules was found to bind and strongly interfere with enzyme-DNA interaction but not with RNA chain elongation. Remarkably, one selected RNA ligand was a specific inhibitor of Saccharomyces cerevisiae Pol II. S. cerevisiae Pol I and Pol III and Pol II from Schizosaccharomyces pombe or wheat germ cells were not affected. Photocross-linking experiments showed that the RNA ligand preferentially interacted with B220, the largest subunit of Pol II and, to a lesser extent, with B150, the second largest subunit. The selected RNA was expressed in yeast cells under the control of a Pol III promoter. Yeast cells that expressed the anti-Pol II aptamer grew normally. However, a cell growth defect was observed when expressing the RNA aptamer in cells having an artificially reduced level of Pol II.

Base Sequence↗
PubMed1994

Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II.

The RNA polymerase II general transcription factor TFIIH is composed of several polypeptides. The observation that the largest subunit of TFIIH is the excision-repair protein XPB/ERCC3 (ref. 1), a helicase implicated in the human DNA-repair disorders xeroderma pigmentosum (XP) and Cockayne's syndrome, suggests a functional link between transcription and DNA repair. To understand the connection between these two cellular processes, we have extensively purified and functionally analysed TFIIH. We find that TFIIH has a dual role, being required for basal transcription of class II genes and for participation in DNA-excision repair. TFIIH is shown to complement three different cell extracts deficient in excision repair: XPB/ERCC3, XPC and XPD/ERCC2. The complementation of XPB and XPD is a consequence of ERCC3 and ERCC2 being integral subunits of TFIIH, whereas complementation of XPC is due to an association of this polypeptide with TFIIH. We found that the general transcription factor IIE negatively modulates the helicase activity of TFIIH through a direct interaction between TFIIE and the ERCC3 subunit of TFIIH.

DNA Helicases↗
PubMed1996

SSN genes that affect transcriptional repression in Saccharomyces cerevisiae encode SIN4, ROX3, and SRB proteins associated with RNA polymerase II.

The RNA polymerase II of Saccharomyces cerevisiae exists in holoenzyme forms containing a complex, known as the mediator, associated with the carboxyl-terminal domain. The mediator includes several SRB proteins and is required for transcriptional activation. Previous work showed that a cyclin-dependent kinase-cyclin pair encoded by SSN3 and SSN8, two members of the SSN suppressor family, are identical to two SRB proteins in the mediator. Here we have identified the remaining SSN genes by cloning and genetic analysis. SSN2 and SSN5 are identical to SRB9 and SRB8, respectively, which encode additional components of the mediator. Genetic evidence implicates the SSN genes in transcriptional repression. Thus, these identities provide genetic insight into mediator and carboxyl-terminal domain function, strongly suggesting a role in mediating transcriptional repression as well as activation. We also show that SSN4 and SSN7 are the same as SIN4 and ROX3, respectively, raising the possibility that these genes also encode mediator proteins.

Base Sequence↗
PubMed1989

Inhibition of in vivo and in vitro transcription by monoclonal antibodies prepared against wheat germ RNA polymerase II that react with the heptapeptide repeat of eukaryotic RNA polymerase II.

Wheat germ RNA polymerase II was used to raise monoclonal antibodies (mAbs) that cross-react with the largest subunit of calf thymus RNA polymerase II. Most of these mAbs were of the IgM isotype and were shown to react with a synthetic peptide containing the consensus sequence for the C-terminal heptapeptide repeat that has been found on the largest subunit of RNA polymerase II from a variety of eukaryotic organisms. A representative mAb (3WG2) was tested for its effect on transcription in both in vitro and in vivo systems. Antibody 3WG2 did not affect the transcription (elongation) of wheat germ RNA polymerase II on denatured calf thymus DNA. When HeLa cell nuclear extracts were preincubated with the mAb, run-off transcription from a promoter that contains a TATA box (the adenovirus-2 major late promoter) and from a promoter that does not contain a TATA box (the murine dihydrofolate reductase gene promoter = dhfr) was inhibited. Transcription from these promoters was also inhibited by the synthetic peptide containing the consensus sequence when it was conjugated to bovine serum albumin. HeLa cell nuclear extract in which the endogenous RNA polymerase II had been inhibited by the specific mAb was used to examine the ability of added mammalian RNA polymerase II that lacks the C-terminal domain to accurately transcribe specific genes. When calf thymus RNA polymerase II that lacked the C-terminal domain was added back to the inhibited extract, a discrete transcript that was initiated correctly was obtained with the adenovirus-2 major late promoter; however, no discrete transcript was observed from the mouse dhfr gene promoter. When injected into Xenopus laevis oocytes, antibody 3WG2 inhibited transcription of the human histone H2b gene (contains a TATA box) and the human U1 small nuclear RNA gene (does not contain a TATA box), but did not inhibit transcription from RNA polymerase I or RNA polymerase III promoters. These results indicate that the C-terminal heptapeptide repeat plays a critical role in promoter-directed transcription, although enzyme that lacks this domain can initiate from some promoters in vitro.

Adenoviridae↗
PubMed1985

A mutation in the largest subunit of RNA polymerase II alters RNA chain elongation in vitro.

An in vitro transcription system which utilized a semisynthetic DNA template (Kadesch, T. R., and Chamberlin, M. J. (1982) J. Biol. Chem. 257, 5286-5295) was developed and used to compare RNA chain elongation by wild type and mutant RNA polymerases II of Drosophila. With this template, all of the active polymerases rapidly initiated RNA chains at synthetic single-stranded sites at the ends of the DNA, and then entered a long (15 to 20 min) period of elongation through duplex regions of template before any measurable termination occurred. A comparison of wild type and mutant polymerase activities during this elongation phase indicated that a mutation to amanitin resistance reduces the rate at which the enzyme elongates transcripts. The reduced elongation rate of the mutant was associated with an altered substrate Km. Because the polymerase II mutation is in the largest enzyme subunit (Greenleaf, A. L. (1983) J. Biol. Chem. 258, 13403-13406), these results demonstrate a functional role for this subunit during RNA chain elongation.

Amanitins↗
PubMed1991

The cloned RNA polymerase II transcription factor IID selects RNA polymerase III to transcribe the human U6 gene in vitro.

Although the human U2 and U6 snRNA genes are transcribed by different RNA polymerases (i.e., RNA polymerases II and III, respectively), their promoters are very similar in structure. Both contain a proximal sequence element (PSE) and an octamer motif-containing enhancer, and these elements are interchangeable between the two promoters. The RNA polymerase III specificity of the U6 promoter is conferred by a single A/T-rich element located around position -25. Mutation of the A/T-rich region converts the U6 promoter into an RNA polymerase II promoter, whereas insertion of the A/T-rich region into the U2 promoter converts that promoter into an RNA polymerase III promoter. We show that this A/T-rich element can be replaced by a number of TATA boxes derived from mRNA promoters transcribed by RNA polymerase II with little effect on RNA polymerase III transcription. Furthermore, the cloned RNA polymerase II transcription factor TFIID both binds to the U6 A/T-rich region and directs accurate RNA polymerase III transcription in vitro. Mutations in the U6 A/T-rich region that convert the U6 promoter into an RNA polymerase II promoter also abolish TFIID binding. Together, these observations suggest that in the human snRNA promoters, unlike in mRNA promoters, binding of TFIID directs the assembly of RNA polymerase III transcription complexes, whereas the lack of TFIID binding results in the assembly of RNA polymerase II snRNA transcription complexes.

Base Sequence↗
PubMed1989

A 7 bp mutation converts a human RNA polymerase II snRNA promoter into an RNA polymerase III promoter.

The human U2 snRNA promoter directs the formation of a specialized RNA polymerase II transcription complex that recognizes the snRNA gene 3' box as a signal for RNA 3' end formation. In contrast, the human U6 promoter is recognized by RNA polymerase III and transcription terminates in a run of Ts. We show that transcription from the U6 promoter is dependent on a sequence similar to the U2 proximal element and on an AT-rich element centered around position -27. Mutation of the AT-rich element induces RNA polymerase II transcription from the U6 promoter, whereas insertion of this element within the U2 promoter converts it into a predominantly RNA polymerase III promoter. The site of transcription termination always correlates with the nature of the transcribing polymerase: the 3' box with RNA polymerase II and a run of Ts with RNA polymerase III. Thus, a single element determines the RNA polymerase specificity of snRNA promoters and hence the site of transcription termination.

Base Composition↗
PubMed1981

[Dependence of RNA synthesis in the cell of Physarum polycephalum on concentration and activity of RNA-polymerase II].

The RNA synthesis detected by incorporation of [H3]uridine chase label proceeds in macroplasmodium and in isolated nuclei throughout the cell cycle of a synchronous myxomycetes culture of Physarum polycephalum, producing two well-shaped peaks at the postmitotic and premitotic steps. Such RNA synthesis is due to the activity of alpha-amanitin-sensitive RNA-polymerase II. Concentrations of RNA-polymerase II at various steps of the cell cycle were determined by alpha-amanitin titration; the specific activity of the enzyme was assayed. The increase in the rate of mRNA synthesis was accompanied by a rise in the concentration and specific activity of the enzyme: for the postmitotic peak, for instance, the corresponding values were about 2-2.5 and 2-4 times as high as minimum values revealed during the cycle. The results obtained are discussed in terms of transcription control in eukaryotic systems.

Amanitins↗
PubMed1996

Increased accommodation of nascent RNA in a product site on RNA polymerase II during arrest.

RNA polymerases encounter specific DNA sites at which RNA chain elongation takes place in the absence of enzyme translocation in a process called discontinuous elongation. For RNA polymerase II, at least some of these sequences also provoke transcriptional arrest where renewed RNA polymerization requires elongation factor SII. Recent elongation models suggest the occupancy of a site within RNA polymerase that accommodates nascent RNA during discontinuous elongation. Here we have probed the extent of nascent RNA extruded from RNA polymerase II as it approaches, encounters, and departs an arrest site. Just upstream of an arrest site, 17-19 nucleotides of the RNA 3'-end are protected from exhaustive digestion by exogenous ribonuclease probes. As RNA is elongated to the arrest site, the enzyme does not translocate and the protected RNA becomes correspondingly larger, up to 27 nucleotides in length. After the enzyme passes the arrest site, the protected RNA is again the 18-nucleotide species typical of an elongation-competent complex. These findings identify an extended RNA product groove in arrested RNA polymerase II that is probably identical to that emptied during SII-activated RNA cleavage, a process required for the resumption of elongation. Unlike Escherichia coli RNA polymerase at a terminator, arrested RNA polymerase II does not release its RNA but can reestablish the normal elongation mode downstream of an arrest site. Discontinuous elongation probably represents a structural change that precedes, but may not be sufficient for, arrest by RNA polymerase II.

Animals↗
PubMed2004

Evolution and distribution of RNA polymerase II regulatory sites from RNA polymerase III dependant mobile Alu elements.

BACKGROUND: The primate-specific Alu elements, which originated 65 million years ago, exist in over a million copies in the human genome. These elements have been involved in genome shuffling and various diseases not only through retrotransposition but also through large scale Alu-Alu mediated recombination. Only a few subfamilies of Alus are currently retropositionally active and show insertion/deletion polymorphisms with associated phenotypes. Retroposition occurs by means of RNA intermediates synthesised by a RNA polymerase III promoter residing in the A-Box and B-Box in these elements. Alus have also been shown to harbour a number of transcription factor binding sites, as well as hormone responsive elements. The distribution of Alus has been shown to be non-random in the human genome and these elements are increasingly being implicated in diverse functions such as transcription, translation, response to stress, nucleosome positioning and imprinting. RESULTS: We conducted a retrospective analysis of putative functional sites, such as the RNA pol III promoter elements, pol II regulatory elements like hormone responsive elements and ligand-activated receptor binding sites, in Alus of various evolutionary ages. We observe a progressive loss of the RNA pol III transcriptional potential with concomitant accumulation of RNA pol II regulatory sites. We also observe a significant over-representation of Alus harboring these sites in promoter regions of signaling and metabolism genes of chromosome 22, when compared to genes of information pathway components, structural and transport proteins. This difference is not so significant between functional categories in the intronic regions of the same genes. CONCLUSIONS: Our study clearly suggests that Alu elements, through retrotransposition, could distribute functional and regulatable promoter elements, which in the course of subsequent selection might be stabilized in the genome. Exaptation of regulatory elements in the preexisting genes through Alus could thus have contributed to evolution of novel regulatory networks in the primate genomes. With such a wide spectrum of regulatory sites present in Alus, it also becomes imperative to screen for variations in these sites in candidate genes, which are otherwise repeat-masked in studies pertaining to identification of predisposition markers.

Alu Elements↗
PubMed2003

Combinatorial control of human RNA polymerase II (RNAP II) pausing and transcript cleavage by transcription factor IIF, hepatitis delta antigen, and stimulatory factor II.

When RNA polymerase II (RNAP II) is forced to stall, elongation complexes (ECs) are observed to leave the active pathway and enter a paused state. Initially, ECs equilibrate between active and paused conformations, but with stalls of a long duration, ECs backtrack and become sensitive to transcript cleavage, which is stimulated by the EC rescue factor stimulatory factor II (TFIIS/SII). In this work, the rates for equilibration between the active and pausing pathways were estimated in the absence of an elongation factor, in the presence of hepatitis delta antigen (HDAg), and in the presence of transcription factor IIF (TFIIF), with or without addition of SII. Rates of equilibration between the active and paused states are not very different in the presence or absence of elongation factors HDAg and TFIIF. SII facilitates escape from stalled ECs by stimulating RNAP II backtracking and transcript cleavage and by increasing rates into and out of the paused EC. TFIIF and SII cooperate to merge the pausing and active pathways, a combinatorial effect not observed with HDAg and SII. In the presence of HDAg and SII, pausing is observed without stimulation of transcript cleavage, indicating that the EC can pause without backtracking beyond the pre-translocated state.

Dose-Response Relationship, Drug↗
PubMed1995

A kinase-cyclin pair in the RNA polymerase II holoenzyme.

The RNA polymerase II holoenzyme consists of RNA polymerase II, a subset of general transcription factors, and regulatory proteins known as SRB proteins. The genes encoding SRB proteins were isolated as suppressors of mutations in the RNA polymerase II carboxy-terminal domain (CTD). The CTD and SRB proteins have been implicated in the response to transcriptional regulators. We report here the isolation of two new SRB genes, SRB10 and SRB11, which encode kinase- and cyclin-like proteins, respectively. Genetic and biochemical evidence indicates that the SRB10 and SRB11 proteins form a kinase-cyclin pair in the holoenzyme. The SRB10/11 kinase is essential for a normal transcriptional response to galactose induction in vivo. Holoenzymes lacking SRB10/11 kinase function are strikingly deficient in CTD phosphorylation. Although defects in the kinase substantially affect transcription in vivo, purified holoenzymes lacking SRB10/11 kinase function do not show defects in defined in vitro transcription systems, suggesting that the factors necessary to elicit the regulatory role of the SRB10/11 kinase are missing in these systems. These results indicate that the SRB10/11 kinase is involved in CTD phosphorylation and suggest that this modification has a role in the response to transcriptional regulators in vivo.

Amino Acid Sequence↗