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 describe an in vitro system that emulates the specific and efficient transcriptional termination associated with the human gastrin gene terminator in vivo. The system involves a dC-tailed DNA template containing the gastrin gene terminator sequence, purified RNA polymerase II, and purified elongation factor TFIIS. In this system, the basal level of termination by RNA polymerase II at the gastrin gene terminator is specifically enhanced by netropsin, an (A + T)-rich minor groove-binding peptide. This enhanced termination is maintained even with TFIIS, which normally suppresses termination at this site. In vitro termination is terminator sequence-specific. Mutant sequences that reduce or abolish termination in vivo show corresponding reductions in activity in the in vitro system. This in vitro emulation of in vivo activities of wild-type and mutant terminators strongly suggests that netropsin and a putative termination factor may share some aspects of their biochemical mechanisms. The general applicability of this system to the study of RNA polymerase II elongation and termination is suggested by the enhancement of termination seen at both the gastrin and human histone H3.3 gene terminators.
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PMID:Netropsin specifically enhances RNA polymerase II termination at terminator sites in vitro. 131 32

Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII.
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PMID:Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II. 150 10

The HIV-1 trans-activator Tat increases the rate of transcription from the HIV-1 LTR promoter through the stem-loop-containing TAR RNA. To analyze the mechanisms of Tat action, a cell-free trans-activation system with no preincubation has been developed. Recombinant Tat specifically increased the level of a long runoff transcript but not a promoter-proximal transcript in a TAR-dependent fashion. These observations and the result of pulse-chase experiments support strongly the hypothesis that Tat enhances the ability of RNA polymerase to elongate over longer distances. Increased levels of the purified cellular factor TFIIF, essential for initiation and also implicated in elongation of transcription, obviated trans-activation by Tat by increasing the basal (Tat-independent) activity. However, another elongation factor, ATN/TFIIS, showed synergistic activation with Tat. An antiserum against a recombinant form of the large subunit of TFIIF (RAP 74) preferentially suppressed the activated level of transcription exerted by Tat. We propose the hypothesis that Tat acts as a processivity factor on RNA polymerase II in an analogous manner to TFIIF.
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PMID:HIV-1 Tat acts as a processivity factor in vitro in conjunction with cellular elongation factors. 155 13

The cDNA for the human elongation factor, TFIIS, has been cloned and expressed in E. coli with the T7 expression system. This 280-amino acid TFIIS protein is shorter by 21 residues than that of the mouse. The missing 21 residues are located in the amino-terminal region, which is not thought to be required for transcriptional stimulation. Apart from this gap, human and mouse proteins reveal 96% overall identity and 98.5% sequence similarity if conservative substitutions are taken into account. The bacterially expressed human protein and the purified calf thymus proteins are indistinguishable in their ability to stimulate transcript elongation by purified RNA polymerase II. Estimation of the native molecular size of the human protein in solution indicates that it exists as a dimer.
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PMID:Cloning, expression and characterization of the human transcription elongation factor, TFIIS. 170 94

The eukaryotic transcriptional factor TFIIS enhances transcript elongation by RNA polymerase II. Here we describe two functional domains in the 280 amino acid human TFIIS protein: residues within positions 100-230 are required for binding to polymerase, and residues 230-280, which form a zinc finger, are required in conjunction with the polymerase binding region for transcriptional stimulation. Interestingly, a mutant TFIIS with only the polymerase binding domain actually inhibits transcription, whereas a mutant in which the polymerase binding and zinc finger domains are separated by an octapeptide is only weakly active. The zinc finger itself has no effect on transcription, but in contrast to the wild-type protein, it binds to oligonucleotides. These findings suggest that TFIIS may interact with RNA polymerase II such that the normally masked zinc finger can specifically contact nucleotides in the transcription elongation zone at a position juxtaposed to the polymerization site.
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PMID:Stimulation of transcript elongation requires both the zinc finger and RNA polymerase II binding domains of human TFIIS. 186 60

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.
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PMID:Factors involved in specific transcription by mammalian RNA polymerase II. Factors IIE and IIF independently interact with RNA polymerase II. 256 9

Highly purified yeast RNA polymerase III ternary complexes were found to possess a hydrolytic chain retracting activity that cleaves nascent RNA from its 3'-OH end. Most of the shortened transcripts were capable of resuming RNA chain elongation, indicating that they remain stably associated with the enzyme-DNA complex. Analysis of the products of cleavage indicated that retraction primarily occurred in dinucleotide increments, but that mononucleotides were also excised at lower frequency. The ribonuclease activity was totally dependent on the presence of a divalent cation and was stimulated by the addition of non-cognate ribonucleotides. The inclusion of ATP in the reaction enhanced both the rate and extent of transcript cleavage. Evidence suggesting that the hydrolytic activity is intrinsic to RNA polymerase III and factor-independent is also presented. Transcript cleavage by RNA polymerase III ternary complexes appears to be more closely related to the intrinsic nucleolytic activity of vaccinia virus RNA polymerase ternary complexes than to TFIIS-dependent cleavage that has been described for RNA polymerase II ternary complexes.
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PMID:Hydrolytic cleavage of nascent RNA in RNA polymerase III ternary transcription complexes. 750 90

In the absence of DNA, purified yeast RNA polymerase II can bind RNA to form a binary complex. RNA in such RNA-RNA polymerase complexes undergoes reactions previously thought to be unique to nascent RNA in ternary complexes with DNA, including TFIIS-dependent cleavage and elongation by 3'-terminal addition of NMP from NTP. Both of these reactions are inhibited by alpha-amanitin. Hence, by several criteria the RNA in binary complexes is bound to the polymerase in a manner quite similar to that in ternary complexes in which the catalytic site for nucleotide addition is positioned at or near the 3'-OH terminus of the RNA. These findings are consistent with a model for the RNA polymerase ternary complex in which the RNA is bound at the 3' terminus through two protein-binding sites located up to 10 nt apart.
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PMID:Complexes of yeast RNA polymerase II and RNA are substrates for TFIIS-induced RNA cleavage. 751 57

Ornithine decarboxylase (ODC) plays an important role in cell proliferation. Its expression is tightly regulated at the mRNA and protein levels and is found to be deregulated in various malignancies. The rapid and dramatic induction of cellular ODC mRNA upon serum addition raised the possibility that a transcriptional attenuation mechanism may be involved in the regulation of ODC gene expression. Using transcription in HeLa nuclear extract and isolated transcription complexes, we have identified two sites of transcription arrest downstream to the transcription start site: Attenuator 1 (Att.1) located at +220, near two repeats of a USF/Myc-Max binding consensus sequence and attenuator 2 (Att.2) located at +1590 near a long stretch of T-residues. The two attenuators exhibit distinct properties as revealed by elongation of briefly initiated and partially purified transcription complexes: Att.1 serves as a transient pause site while arrest at Att.2 is more prolonged. The arrest at both attenuators is modulated by the general elongation factor TFIIS. In a promoter independent transcription system, using partially purified RNA polymerase II, only Att.2 was recognized efficiently. This suggests that the recognition of Att.2 is an intrinsic property of the polymerase while Att.1 recognition has to be facilitated by an auxiliary factor/s.
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PMID:Transcription elongation of the murine ornithine decarboxylase (ODC) gene is regulated in vitro at two downstream elements by different attenuation mechanisms. 753 63

TFIIS is a transcription elongation factor that binds to RNA polymerase II and allows it to transcribe through a variety of transcriptional blockages by inducing cleavage near the 3' end of the nascent transcript. Although this cleavage reaction plays a key role in the process of reactivation of transcription by TFIIS, the exact mechanism by which TFIIS promotes readthrough by RNA polymerase II is not completely understood. We therefore undertook a systematic mutagenesis of the C-terminal half of TFIIS (delta TFIIS) to evaluate the contribution of charged residues in this region to induce transcript cleavage and promote readthrough in vitro. Twenty-two delta TFIIS alanine-scanning mutants were constructed by substitution of alanine for each amino acid in clusters of charged residues in the C-terminal half of HeLa TFIIS. The ability to induce transcript cleavage and readthrough of these mutants was tested in vitro using RNA polymerase II ternary elongation complexes arrested at a block to elongation. This alanine-scanning mutagenesis analysis allowed the identification of regions or residues important for the activity of TFIIS. Many of the mutants were reduced alike in both cleavage and readthrough activities. However, in several cases there was no simple correlation between these activity reductions.
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PMID:Alanine-scanning mutagenesis of human transcript elongation factor TFIIS. 757 53


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