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)

Elongation factor SII (also known as TFIIS) is an RNA polymerase II binding protein that allows bypass of template arrest sites by activating a nascent RNA cleavage reaction. Here we show that SII contacts the 3'-end of nascent RNA within an RNA polymerase II elongation complex as detected by photoaffinity labeling. Photocross-linking was dependent upon the presence of SII, incorporation of 4-thio-UMP into RNA, and irradiation and was sensitive to treatment by RNase and proteinase. A transcriptionally active mutant of SII lacking the first 130 amino acids was also cross-linked to the nascent RNA, but SII from Saccharomyces cerevisiae, which is inactive in concert with mammalian RNA polymerase II, failed to become photoaffinity labeled. SII-RNA contact was not detected after a labeled oligoribonucleotide was released from the complex by nascent RNA cleavage, demonstrating that this interaction takes place between elongation complex-associated but not free RNA. This shows that the 3'-end of RNA is near the SII binding site on RNA polymerase II and suggests that SII may activate the intrinsic RNA hydrolysis activity by positioning the transcript in the enzyme's active site.
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PMID:Elongation factor SII contacts the 3'-end of RNA in the RNA polymerase II elongation complex. 879 87

A relatively simple subset of general transcription factors is sufficient for transcript initiation by RNA polymerase II. However, a recently identified "holoenzyme" contains additional accessory proteins required for mediating signals from some activators (Y-J. Kim et al., 1994, Cell 77, 599-608; A. Koleske and R. Young, 1994, Nature 368, 466-469). By immobilizing RNA polymerase II and associated proteins (RAPs) from a transcriptionally active yeast extract, we have identified a novel collection of proteins distinct from those found in the holoenzyme. The eluted RAP fraction did not contain the holoenzyme components Srb2,4,5 + 6p, Gal11p, or Sug1p, but did include the known transcription factors TFIIB and TFIIS and the three subunits of yeast TFIIF (Ssu71p/Tfg1p, Tfg2p, and Anc1p/Tfg3p). Also isolated as RAPs are two proteins (Cdc73p and Paf1p) with interesting connections to gene expression. Mutations in CDC73 and PAF1 affect cell growth and the abundance of transcripts from a subset of yeast genes (X. Shi et al., Mol. Cell. Biol., 1996 16, 669-676). The RAP fraction may therefore define one or more functional forms of RNA polymerase II distinct from the activator-mediating holoenzyme.
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PMID:A novel collection of accessory factors associated with yeast RNA polymerase II. 881 38

Transcription elongation by RNA polymerase II is regulated by the general elongation factor TFIIS. This factor stimulates RNA polymerase II to transcribe through regions of DNA that promote the formation of stalled ternary complexes. Limited proteolytic digestion showed that yeast TFIIS is composed of three structural domains, termed I, II, and III. The two C-terminal domains (II and III) are required for transcription activity. The structure of domain III has been solved previously by using NMR spectroscopy. Here, we report the NMR-derived structure of domain II: a three-helix bundle built around a hydrophobic core composed largely of three tyrosines protruding from one face of the C-terminal helix. The arrangement of known inactivating mutations of TFIIS suggests that two surfaces of domain II are critical for transcription activity.
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PMID:Elongation factor TFIIS contains three structural domains: solution structure of domain II. 885 25

We have reported previously the isolation and genetic characterization of mutations in the gene encoding the largest subunit of yeast RNA polymerase II (RNAPII), which lead to 6-azauracil (6AU)-sensitive growth. It was suggested that these mutations affect the functional interaction between RNAPII and transcription-elongation factor TFIIS because the 6AU-sensitive phenotype of the mutant strains was similar to that of a strain defective in the production of TFIIS and can be suppressed by increasing the dosage of the yeast TFIIS-encoding gene, PPR2, RNAPIIs were purified and characterized from two independent 6AU-sensitive yeast mutants and from wild-type (wt) cells. In vitro, in the absence of TFIIS, the purified wt polymerase and the two mutant polymerases showed similar specific activity in polymerization, readthrough at intrinsic transcriptional arrest sites and nascent RNA cleavage. In contrast to the wt polymerase, both mutant polymerases were not stimulated by the addition of a 3-fold molar excess of TFIIS in assays of promoter-independent transcription, readthrough or cleavage. However, stimulation of the ability of the mutant RNAPIIs to cleave nascent RNA and to read through intrinsic arrest sites was observed at TFIIS:RNAPII molar ratios greater than 600:1. Consistent with these findings, the binding affinity of the mutant polymerases for TFIIS was found to be reduced by more than 50-fold compared with that of the wt enzyme. These studies demonstrate that TFIIS has an important role in the regulation of transcription by yeast RNAPII and identify a possible binding site for TFIIS on RNAPII.
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PMID:In vitro characterization of mutant yeast RNA polymerase II with reduced binding for elongation factor TFIIS. 887 73

Efficient transcription elongation by RNA polymerase I (Pol I) requires a specific Pol I-associated factor, termed TIF-IC. Here we show that TFIIS, a factor that has previously been shown to promote read-through past many types of blocks to elongation by RNA polymerase II, also enhances Pol I-directed transcription elongation. In a reconstituted transcription system containing purified proteins, TFIIS stimulates Pol I transcription by increasing the overall rate of RNA chain elongation. As with Pol II, ternary Pol I complexes cleave the 3' end of the nascent transcripts in response to TFIIS. The truncated RNAs remain bound to the template, are subject to pyrophosphorolysis, and can be chased into longer transcripts. Moreover, we show by immunoprecipitation and specific affinity chromatography that TFIIS physically interacts with Pol I. The results suggest that nascent transcript cleavage by TFIIS or a TFIIS-related factor may be a general mechanism by which both Pol I and Pol II can bypass transcriptional impediments.
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PMID:TFIIS binds to mouse RNA polymerase I and stimulates transcript elongation and hydrolytic cleavage of nascent rRNA. 887 42

Fidelity of DNA and protein synthesis is regulated by a proofreading mechanism but function of a similar mechanism during RNA synthesis has not been demonstrated. Analysis of transcriptional fidelity and its control has been hampered by the necessity to employ complex DNA templates requiring either a promoter and initiation factors or 3'-extended templates. To circumvent this difficulty, we have created an RNA-DNA dumbbell template that can be recognized as a template-primer and extended by RNA polymerase II. By employing this system, we demonstrate that RNA polymerase II can misincorporate a nucleotide and carry out template-dependent elongation at the mispaired end. The transcripts containing misincorporated residues can be cleaved by the very slow 3'-->5' ribonuclease activity of the RNA polymerase II, but enhancement of this activity by the elongation factor TFIIS generates RNA with a high degree of fidelity. This enhanced preferential cleavage of misincorporated transcripts suggests an important role for TFIIS in maintaining transcriptional fidelity.
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PMID:Fidelity of RNA polymerase II transcription controlled by elongation factor TFIIS. 894 93

The products of the yeast CDC73 and PAF1 genes were originally identified as RNA polymerase II-associated proteins. Paf1p is a nuclear protein important for cell growth and transcriptional regulation of a subset of yeast genes. In this study we demonstrate that the product of CDC73 is a nuclear protein that interacts directly with purified RNA polymerase II in vitro. Deletion of CDC73 confers a temperature-sensitive phenotype. Combination of the cdc73 mutation with the more severe paf1 mutation does not result in an enhanced phenotype, indicating that the two proteins may function in the same cellular processes. To determine the relationship between Cdc73p and Paf1p and the recently described holoenzyme form of RNA polymerase II, we created yeast strains containing glutathione S-transferase (GST)-tagged forms of CDC73, PAF1, and TFG2 functionally replacing the chromosomal copies of the genes. Isolation of GST-tagged Cdc73p and Paf1p complexes has revealed a unique form of RNA polymerase II that contains both Cdc73p and Paf1p but lacks the Srbps found in the holoenzyme. The Cdc73p-Paf1p-RNA polymerase II-containing complex also includes Gal11p, and the general initiation factors TFIIB and TFIIF, but lacks TBP, TFIIH, and transcription elongation factor TFIIS as well as the Srbps. The Srbp-containing holoenzyme does not include either Paf1p or Cdc73p, demonstrating that these two forms of RNA polymerase II are distinct. In confirmation of the hypothesis that the two forms coexist in yeast cells, we found that a TFIIF-containing complex isolated via the GST-tagged Tfg2p construct contains both (i) the Srbps and (ii) Cdc73p and Paf1p. The Srbps and Cdc73p-Paf1p therefore appear to define two complexes with partially redundant, essential functions in the yeast cell. Using the technique of differential display, we have identified several genes whose transcripts require Cdc73p and/or Paf1p for normal levels of expression. Our analysis suggests that there are multiple RNA polymerase II-containing complexes involved in the expression of different classes of protein-coding genes.
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PMID:Cdc73p and Paf1p are found in a novel RNA polymerase II-containing complex distinct from the Srbp-containing holoenzyme. 903 43

Stalled Xenopus RNA polymerase I (pol I) elongation complexes bearing a 52-nucleotide RNA were prepared by promoter-initiated transcription in the absence of UTP. When such complexes were isolated and incubated in the presence of Mg2+, the associated RNA was shortened from the 3'-end, and mono- and dinucleotides were released. Shortened transcripts were still associated with the DNA and were quantitatively reelongated upon addition of NTPs. The cleavage activity could be removed from the pol I-ternary complex with buffers containing 0.25% Sarkosyl. These findings indicate that a factor with characteristics similar to elongation factor TFIIS is associated with the pol I elongation complex. However, addition of recombinant Xenopus TFIIS to Sarkosyl-washed pol I elongation complexes had no effect, whereas it showed the expected effects in control reactions with identically prepared pol II elongation complexes. The results thus suggest the existence of a pol I-specific cleavage/elongation factor. I also report the sequence of a novel type of Xenopus TFIIS. The predicted amino acid sequences of the present and previously identified Xenopus TFIIS are less than 65% conserved. Thus, like mammalian species, Xenopus has at least two highly divergent forms of TFIIS.
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PMID:Transcript cleavage in an RNA polymerase I elongation complex. Evidence for a dissociable activity similar to but distinct from TFIIS. 908 31

The role of yeast RNA polymerase II (pol II) subunit RPB9 in transcript elongation was investigated by examining the biochemical properties of pol II lacking RPB9 (pol IIDelta9). The maximal rate of chain elongation was nearly identical for pol II and pol IIDelta9. By contrast, pol IIDelta9 elongated more efficiently through DNA sequences that signal the elongation complex to pause or arrest. The addition of purified recombinant RPB9 to pol IIDelta9 restored the elongation properties of the mutant polymerase to those of the wild-type enzyme. Arrested pol IIDelta9 complexes were refractory to levels of TFIIS that promoted maximal read-through with pol II. However, both pol II and pol IIDelta9 complexes stimulated with TFIIS undergo transcript cleavage, confirming that transcript cleavage and read-through activities can be uncoupled. Our observations suggest that both TFIIS and RPB9 are required to stimulate the release of RNA polymerase II from the arrested state.
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PMID:Transcription elongation through DNA arrest sites. A multistep process involving both RNA polymerase II subunit RPB9 and TFIIS. 916 40

Affinity chromatography on columns containing the immobilized monomeric transcriptional elongation factor TFIIS or the essential large subunit, Elongin A, of the trimeric elongation factor, Elongin, was used to purify a human RNA polymerase II holoenzyme from HeLa whole cell extract. This holoenzyme contained nearstoichiometric amounts of all the general transcription factors, TFIIB, TFIID (TBP + TAFIIs), TFIIE, TFIIF, and TFIIH, required to accurately initiate transcription in vitro at the adenovirus major late promoter. It behaved as a large complex, slightly smaller than 70 S ribosomes, during gel filtration chromatography, and contained nearly half the TFIID that was present in the extract used for the affinity chromatography. It also contained the cyclin-dependent kinase CDK8, a human homologue of the Saccharomyces cerevisiae holoenzyme subunit SRB10, and many other polypeptides. Efficient interaction of holoenzyme with TFIIS or Elongin A required only the amino-terminal region of either protein. These regions are similar in amino acid sequence but dispensable for TFIIS or Elongin to regulate elongation in vitro by highly purified RNA polymerase II. The transcriptional activators GAL4-VP16 and GAL4-Sp1 activated transcription in vitro by purified holoenzyme in the absence of any additional factors.
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PMID:Interaction of elongation factors TFIIS and elongin A with a human RNA polymerase II holoenzyme capable of promoter-specific initiation and responsive to transcriptional activators. 930 22


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