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)

DNA sequences that arrest transcription by either eukaryotic RNA polymerase II or Escherichia coli RNA polymerase have been identified previously. Elongation factors SII and GreB are RNA polymerase-binding proteins that enable readthrough of arrest sites by these enzymes, respectively. This functional similarity has led to general models of elongation applicable to both eukaryotic and prokaryotic enzymes. Here we have transcribed with phage and bacterial RNA polymerases, a human DNA sequence previously defined as an arrest site for RNA polymerase II. The phage and bacterial enzymes both respond efficiently to the arrest signal in vitro at limiting levels of nucleoside triphosphates. The E. coli polymerase remains in a template-engaged complex for many hours, can be isolated, and is potentially active. The enzyme displays a relatively slow first-order loss of elongation competence as it dwells at the arrest site. Bacterial RNA polymerase arrested at the human site is reactivated by GreB in the same way that RNA polymerase II arrested at this site is stimulated by SII. Very efficient readthrough can be achieved by phage, bacterial, and eukaryotic RNA polymerases in the absence of elongation factors if 5-Br-UTP is substituted for UTP. These findings provide additional and direct evidence for functional similarity between prokaryotic and eukaryotic transcription elongation and readthrough mechanisms.
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PMID:Recognition of a human arrest site is conserved between RNA polymerase II and prokaryotic RNA polymerases. 964 44

Elongation factor SII interacts with RNA polymerase II and enables it to transcribe through arrest sites in vitro. The set of genes dependent upon SII function in vivo and the effects on RNA levels of mutations in different components of the elongation machinery are poorly understood. Using yeast lacking SII and bearing a conditional allele of RPB2, the gene encoding the second largest subunit of RNA polymerase II, we describe a genetic interaction between SII and RPB2. An SII gene disruption or the rpb2-10 mutation, which yields an arrest-prone enzyme in vitro, confers sensitivity to 6-azauracil (6AU), a drug that depresses cellular nucleoside triphosphates. Cells with both mutations had reduced levels of total poly(A)+ RNA and specific mRNAs and displayed a synergistic level of drug hypersensitivity. In cells in which the SII gene was inactivated, rpb2-10 became dominant, as if template-associated mutant RNA polymerase II hindered the ability of wild-type polymerase to transcribe. Interestingly, while 6AU depressed RNA levels in both wild-type and mutant cells, wild-type cells reestablished normal RNA levels, whereas double-mutant cells could not. This work shows the importance of an optimally functioning elongation machinery for in vivo RNA synthesis and identifies an initial set of candidate genes with which SII-dependent transcription can be studied.
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PMID:Mutations in RNA polymerase II and elongation factor SII severely reduce mRNA levels in Saccharomyces cerevisiae. 974 94

On 5'-template strand protruding templates, promoter-initiated run-off transcription by RNA polymerase II generates discrete, 15-16-nucleotide (nt) longer than expected products whose production is abrogated by elongation factor SII (Parsons, M. A., Sinden, R. R., and Izban, M. G. (1998) J. Biol. Chem. 273, 26998-27008). We demonstrate that template terminal complexes produce these RNAs and that transcript extension is a general and salt-sensitive (250 mM) feature of run-off transcription. On 5'-overhung templates the extended run-off transcripts appear to be retained within an RNA-DNA-enzyme ternary complex, and SII facilitates resumption of transcript elongation via a dinucleotide truncation intermediate. Moreover, on one of the 5-overhung templates, the initially extended complexes spontaneously resumed transcript extension and were uniquely resistant to salt (250 mM) challenge. However, SII did not facilitate this long distance extension on all template ends. Run-off transcripts on a blunt-ended template were initially extended by 2-11 nt (roughly in 2-nt increments); SII addition either before or after extension resulted in the accumulation of a 4-5-nt extension product. Based on these findings, we propose that the initial and continuously extended RNAs reflect intermediates and successful completion of template end-to-end transposition (template switching) by RNA polymerase II, respectively. Both the template end sequence and structure influenced the success of such an event.
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PMID:Template end-to-end transposition by RNA polymerase II. 975 51

We have characterized the properties of immunopurified transcription complexes arrested at a specifically located cyclobutane pyrimidine dimer (CPD) using enzymatic probes and an in vitro transcription system with purified RNA polymerase II (RNAP II) and initiation factors. To help understand how RNAP II distinguishes between a natural impediment and a lesion in the DNA to initiate a repair event, we have compared the conformation of RNAP II complexes arrested at a CPD with complexes arrested at a naturally occurring elongation impediment. The footprint of RNAP II arrested at a CPD, using exonuclease III and T4 DNA polymerase's 3'-->5' exonuclease, covers approximately 35 base pairs and is asymmetrically located around the dimer. A similar footprint is observed when RNAP II is arrested at the human histone H3.3 arrest site. Addition of elongation factor SII to RNAP II arrested at a CPD produced shortened transcripts of discrete lengths up to 25 nucleotides shorter than those seen without SII. After addition of photolyase and exposure to visible light, some of the transcripts could be reelongated beyond the dimer, suggesting that SII-mediated transcript cleavage accompanied significant RNAP II backup, thereby providing access of the repair enzyme to the arresting CPD.
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PMID:Structural characterization of RNA polymerase II complexes arrested by a cyclobutane pyrimidine dimer in the transcribed strand of template DNA. 1044 84

RNA chain elongation by RNA polymerase II (pol II) is a complex and regulated process which is coordinated with capping, splicing, and polyadenylation of the primary transcript. Numerous elongation factors that enable pol II to transcribe faster and/or more efficiently have been purified. SII is one such factor. It helps pol II bypass specific blocks to elongation that are encountered during transcript elongation. SII was first identified biochemically on the basis of its ability to enable pol II to synthesize long transcripts. ((1)) Both the high resolution structure of SII and the details of its novel mechanism of action have been refined through mutagenesis and sophisticated in vitro assays. SII engages transcribing pol II and assists it in bypassing blocks to elongation by stimulating a cryptic, nascent RNA cleavage activity intrinsic to RNA polymerase. The nuclease activity can also result in removal of misincorporated bases from RNA. Molecular genetic experiments in yeast suggest that SII is generally involved in mRNA synthesis in vivo and that it is one type of a growing collection of elongation factors that regulate pol II. In vertebrates, a family of related SII genes has been identified; some of its members are expressed in a tissue-specific manner. The principal challenge now is to understand the isoform-specific functional differences and the biology of regulation exerted by the SII family of proteins on target genes, particularly in multicellular organisms.
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PMID:Transcription elongation factor SII. 1072 30

IMP dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo synthesis of guanine nucleotides. It is a target of therapeutically useful drugs and is implicated in the regulation of cell growth rate. In the yeast Saccharomyces cerevisiae, mutations in components of the RNA polymerase II (Pol II) transcription elongation machinery confer increased sensitivity to a drug that inhibits IMPDH, 6-azauracil (6AU), by a mechanism that is poorly understood. This phenotype is thought to reflect the need for an optimally functioning transcription machinery under conditions of lowered intracellular GTP levels. Here we show that in response to the application of IMPDH inhibitors such as 6AU, wild-type yeast strains induce transcription of PUR5, one of four genes encoding IMPDH-related enzymes. Yeast elongation mutants sensitive to 6AU, such as those with a disrupted gene encoding elongation factor SII or those containing amino acid substitutions in Pol II subunits, are defective in PUR5 induction. The inability to fully induce PUR5 correlates with mutations that effect transcription elongation since 6AU-sensitive strains deleted for genes not related to transcription elongation are competent to induce PUR5. DNA encompassing the PUR5 promoter and 5' untranslated region supports 6AU induction of a luciferase reporter gene in wild-type cells. Thus, yeast sense and respond to nucleotide depletion via a mechanism of transcriptional induction that restores nucleotides to levels required for normal growth. An optimally functioning elongation machinery is critical for this response.
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PMID:Saccharomyces cerevisiae transcription elongation mutants are defective in PUR5 induction in response to nucleotide depletion. 1100 40

Gre proteins of prokaryotes, and SII proteins of eukaryotes and archaea, are transcription elongation factors that promote an endogenous transcript cleavage activity of RNA polymerases; this process promotes elongation through obstructive regions of DNA, including transcription pauses that act as sites of genetic regulation. We show that a regulatory pause in the early part of the late gene operon of bacteriophage lambda is subject to such cleavage and resynthesis. In cells lacking the cleavage factors GreA and GreB, the pause is prolonged, and RNA polymerase occupies a variant position at the pause site. Furthermore, GreA and GreB are required to mediate efficient function of the lambda gene Q antiterminator at this site. Thus, cleavage factors are necessary for the natural progression of RNA polymerase in vivo.
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PMID:Function of transcription cleavage factors GreA and GreB at a regulatory pause site. 1116 2

Nucleotide excision repair is the major pathway responsible for removing UV-induced DNA damage, and is therefore essential for cell survival following exposure to UV radiation. In this report, we have assessed the contributions of some components of the RNA polymerase II (Pol II) transcription machinery to UV resistance in Saccharomyces cerevisiae. Deletion of the gene encoding the Pol II elongation factor TFIIS (SII) resulted in enhanced UV sensitivity, but only in the absence of global genome repair dependent on the RAD7 and RAD16 genes, a result seen previously with deletions of RAD26 and RAD28, yeast homologs of the human Cockayne syndrome genes CSB and CSA, respectively. A RAD7/16-dependent reduction in survival after UV irradiation was also seen in the presence of mutations in RNA Pol II that confer a defect in its response to SII, as well as with other mutations which reside in regions of the largest subunit of Pol II not involved in SII interactions. Indeed, an increase in UV sensitivity was achieved by simply decreasing the steadystate level of RNA Pol II. Truncation of the C-terminal domain and other RNA Pol II mutations conferred sensitivity to the ribonucleotide reductase inhibitor hydroxyurea and induction of RNR1 and RNR2 mRNAs after UV irradiation was attenuated in these mutant cells. That UV sensitivity can be a consequence of mutations in the RNA Pol II machinery in yeast cells suggests that alterations in transcriptional programs could underlie some of the pathophysiological defects seen in the human disease Cockayne syndrome.
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PMID:A compromised yeast RNA polymerase II enhances UV sensitivity in the absence of global genome nucleotide excision repair. 1125 32

TFIIF, ELL, and Elongin belong to a class of RNA polymerase II transcription factors that function similarly to activate the rate of elongation by suppressing transient pausing by polymerase at many sites along DNA templates. SII is a functionally distinct RNA polymerase II elongation factor that promotes elongation by reactivating arrested polymerase. Studies of the mechanism of SII action have shown (i) that arrest of RNA polymerase II results from irreversible displacement of the 3'-end of the nascent transcript from the polymerase catalytic site and (ii) that SII reactivates arrested polymerase by inducing endonucleolytic cleavage of the nascent transcript by the polymerase catalytic site thereby creating a new transcript 3'-end that is properly aligned with the catalytic site and can be extended. SII also induces nascent transcript cleavage by paused but non-arrested RNA polymerase II elongation intermediates, leading to the proposal that pausing may result from reversible displacement of the 3'-end of nascent transcripts from the polymerase catalytic site. On the basis of evidence consistent with the model that TFIIF, ELL, and Elongin suppress pausing by preventing displacement of the 3'-end of the nascent transcript from the polymerase catalytic site, we investigated the possibility of cross-talk between SII and transcription factors TFIIF, ELL, and Elongin. These studies led to the discovery that TFIIF, ELL, and Elongin are all capable of inhibiting SII-induced nascent transcript cleavage by non-arrested RNA polymerase II elongation intermediates. Here we present these findings, which bring to light a novel activity associated with TFIIF, ELL, and Elongin and suggest that these transcription factors may expedite elongation not only by increasing the forward rate of nucleotide addition by RNA polymerase II, but also by inhibiting SII-induced nascent transcript cleavage by non-arrested RNA polymerase II elongation intermediates.
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PMID:Transcription factors TFIIF, ELL, and Elongin negatively regulate SII-induced nascent transcript cleavage by non-arrested RNA polymerase II elongation intermediates. 1125 17

In vitro, transcript elongation by RNA polymerase II is impeded by DNA sequences, DNA-bound proteins, and small ligands. Transcription elongation factor SII (TFIIS) assists RNA polymerase II to transcribe through these obstacles. There is however, little direct evidence that SII-responsive arrest sites function in living cells nor that SII facilitates readthrough in vivo. Saccharomyces cerevisiae strains lacking elongation factor SII and/or containing a point mutation in the second largest subunit of RNA polymerase II, which slows the enzyme's RNA elongation rate, grow slowly and have defects in mRNA metabolism, particularly in the presence of nucleotide-depleting drugs. Here we have examined transcriptional induction in strains lacking SII or containing the slow polymerase mutation. Both mutants and a combined double mutant were defective in induction of GAL1 and ENA1. This was not due to an increase in mRNA degradation and was independent of any drug treatment, although treatment with the nucleotide-depleting drug 6-azauracil exacerbated the effect preferentially in the mutants. These data are consistent with mutants in the Elongator complex, which show slow inductive responses. When a potent in vitro arrest site was transcribed in these strains, there was no perceptible effect upon mRNA accumulation. These data suggest that an alternative elongation surveillance mechanism exists in vivo to overcome arrest.
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PMID:Analysis of gene induction and arrest site transcription in yeast with mutations in the transcription elongation machinery. 1127 87


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