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)

Iws1 has been implicated in transcriptional elongation by interaction with RNA polymerase II (RNAP II) and elongation factor Spt6 in budding yeast Saccharomyces cerevisiae, and association with transcription factor TFIIS in mammalian cells, but its role in controlling cell growth and proliferation remains unknown. Here we report that the human homolog of Iws1, hIws1, physically interacts with protein arginine methyltransferases PRMT5 which methylates elongation factor Spt5 and regulates its interaction with RNA polymerase II. Gene-specific silencing of hIws1 by RNA interference reveals that hIws1 is essential for cell viability. GFP fusion protein expression approaches demonstrate that the hIws1 protein is located in the nucleus, subsequently, two regions harbored within the hIws1 protein are demonstrated to contain nuclear localization signals (NLSs). In addition, mouse homolog of hiws1 is found to express ubiquitously in various tissues.
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PMID:A putative transcriptional elongation factor hIws1 is essential for mammalian cell proliferation. 1718 35

RNA polymerase II (RNAP II) is responsible for transcribing all messenger RNAs in eukaryotic cells during a highly regulated process that is conserved from yeast to human, and that serves as a central control point for cellular function. Here we investigate the transcription dynamics of single RNAP II molecules from Saccharomyces cerevisiae against force and in the presence and absence of TFIIS, a transcription elongation factor known to increase transcription through nucleosomal barriers. Using a single-molecule dual-trap optical-tweezers assay combined with a novel method to enrich for active complexes, we found that the response of RNAP II to a hindering force is entirely determined by enzyme backtracking. Surprisingly, RNAP II molecules ceased to transcribe and were unable to recover from backtracks at a force of 7.5 +/- 2 pN, only one-third of the force determined for Escherichia coli RNAP. We show that backtrack pause durations follow a t(-3/2) power law, implying that during backtracking RNAP II diffuses in discrete base-pair steps, and indicating that backtracks may account for most of RNAP II pauses. Significantly, addition of TFIIS rescued backtracked enzymes and allowed transcription to proceed up to a force of 16.9 +/- 3.4 pN. Taken together, these results describe a regulatory mechanism of transcription elongation in eukaryotes by which transcription factors modify the mechanical performance of RNAP II, allowing it to operate against higher loads.
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PMID:Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner. 1736 Nov 30

The blockage of transcription elongation by RNA polymerase II (RNAPII) at DNA lesions on the transcribed strand is a serious challenge to accurate transcription. Transcription-coupled DNA repair (TCR), which is assumed to be initiated by the blockage of transcription, rapidly removes lesions on the transcribed strand of expressed genes and allows the resumption of transcription. Although helix-distorting bulky damage such as a cyclobutane pyrimidine dimer is known to block transcription elongation and to be repaired by TCR, it is not clear whether oxidative DNA lesions are repaired by TCR. First, we examined whether transcription elongation by RNAPII is stalled at sites of 2-hydroxyadenine (2-OH-A), 8-oxoadenine (8-oxoA), 8-oxoguanine (8-oxoG), or thymine glycol (Tg) on the transcribed strand. Our results indicate that RNAPII incorporated nucleotides opposite the lesions and then stalled. In addition, we found that transcription elongation factor TFIIS (SII) enabled RNAPII to bypass 8-oxoG but not the other types of damage, while transcription initiation and elongation factor TFIIF did not bypass 8-oxoG. These results suggest that SII is important for preventing cellular death due to oxidative DNA damage, assisting RNAPII to bypass 8-oxoG.
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PMID:RNA polymerase II bypasses 8-oxoguanine in the presence of transcription elongation factor TFIIS. 1737 14

The transcription elongation factor S-II, also designated TFIIS, stimulates the nascent transcript cleavage activity intrinsic to RNA polymerase II. Rpb9, a small subunit of RNA polymerase II, enhances the cleavage stimulation activity of S-II. Here, we investigated the role of nascent transcript cleavage stimulation activity on the maintenance of transcriptional fidelity in yeast. In yeast, S-II is encoded by the DST1 gene. Disruption of the DST1 gene decreased transcriptional fidelity in cells. Mutations in the DST1 gene that reduce the S-II cleavage stimulation activity led to decreased transcriptional fidelity in cells. A disruption mutant of the RPB9 gene also had decreased transcriptional fidelity. Expression of mutant Rpb9 proteins that are unable to enhance the S-II cleavage stimulation activity failed to restore the phenotype. These results suggest that both S-II and Rpb9 maintain transcriptional fidelity by stimulating the cleavage activity intrinsic to RNA polymerase II. Also, a DST1 and RPB9 double mutant had more severe transcriptional fidelity defect compared with the DST1 gene deletion mutant, suggesting that Rpb9 maintains transcriptional fidelity via two mechanisms, enhancement of S-II dependent cleavage stimulation and S-II independent function(s).
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PMID:Stimulation of RNA polymerase II transcript cleavage activity contributes to maintain transcriptional fidelity in yeast. 1753 46

When nucleoside triphosphate (NTP) substrates and alpha-amanitin are added to a human RNA polymerase II elongation complex simultaneously, the reaction becomes stalled in the core of the bond synthesis mechanism. The mode of stalling is influenced by NTP substrates at the active site and at downstream sites and by transcription factor IIF (TFIIF) and TFIIS. NTP substrates templated at i+2, i+3, and i+4 downstream DNA sites can reverse the previously stable binding of an NTP loaded at the i+1 substrate site. Deoxy-(d)NTPs and NDPs (nucleoside diphosphates) do not substitute for NTPs at the i+2 and i+3 positions (considered together) or the i+4, i+5, and i+6 positions (considered together). The mode of stalling is altered by changing the number of downstream template sites that are accurately occupied by NTPs and by changing NTP concentration. In the presence of the translocation blocker alpha-amanitin, a steady state condition is established in which RNA polymerase II stably loads an NTP substrate at i+1 and forms a phosphodiester bond but cannot rapidly complete bond synthesis by releasing pyrophosphate. These observations support a role for incoming NTP substrates in stimulating translocation; results appear inconsistent with the secondary pore being the sole route of NTP entry for human RNA polymerase II, and results indicate mechanisms of dynamic error avoidance and error correction during rapid RNA synthesis.
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PMID:A tunable ratchet driving human RNA polymerase II translocation adjusted by accurately templated nucleoside triphosphates loaded at downstream sites and by elongation factors. 1787 40

In this article, we provide direct evidence that the evolutionarily conserved transcription elongation factor TFIIS functions during preinitiation complex assembly. First, we identified TFIIS in a mass spectrometric screen of RNA polymerase II (Pol II) preinitiation complexes (PICs). Second, we show that the association of TFIIS with a promoter depends on functional PIC components including Mediator and the SAGA complex. Third, we demonstrate that TFIIS is required for efficient formation of active PICs. Using truncation mutants of TFIIS, we find that the Pol II-binding domain is the minimal domain necessary to stimulate PIC assembly. However, efficient formation of active PICs requires both the Pol II-binding domain and the poorly understood N-terminal domain. Importantly, Domain III, which is required for the elongation function of TFIIS, is dispensable during PIC assembly. The results demonstrate that TFIIS is a PIC component that is required for efficient formation and/or stability of the complex.
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PMID:The transcription elongation factor TFIIS is a component of RNA polymerase II preinitiation complexes. 1791 84

Positive transcription elongation factor b (P-TEFb) is the major metazoan RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) Ser2 kinase, and its activity is believed to promote productive elongation and coupled RNA processing. Here, we demonstrate that P-TEFb is critical for the transition of Pol II into a mature transcription elongation complex in vivo. Within 3 min following P-TEFb inhibition, most polymerases were restricted to within 150 bp of the transcription initiation site of the active Drosophila melanogaster Hsp70 gene, and live-cell imaging demonstrated that these polymerases were stably associated. Polymerases already productively elongating at the time of P-TEFb inhibition, however, proceeded with elongation in the absence of active P-TEFb and cleared from the Hsp70 gene. Strikingly, all transcription factors tested (P-TEFb, Spt5, Spt6, and TFIIS) and RNA-processing factor CstF50 exited the body of the gene with kinetics indistinguishable from that of Pol II. An analysis of the phosphorylation state of Pol II upon the inhibition of P-TEFb also revealed no detectable CTD Ser2 phosphatase activity upstream of the Hsp70 polyadenylation site. In the continued presence of P-TEFb inhibitor, Pol II levels across the gene eventually recovered.
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PMID:P-TEFb is critical for the maturation of RNA polymerase II into productive elongation in vivo. 1807 Sep 27

In this issue, Kuhn et al. (2007) report the complete structure of the 14-subunit yeast RNA polymerase (Pol) I enzyme at 12 A resolution using cryo-electron microscopy (cryo-EM). Their study reveals that three subunits of Pol I perform functions in transcription elongation that are outsourced to the transcription factors TFIIF and TFIIS in the analogous Pol II transcription system.
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PMID:RNA polymerase I: a multifunctional molecular machine. 1816 37

The encounter of elongating RNA polymerase II (RNAPIIo) with DNA lesions has severe consequences for the cell as this event provides a strong signal for P53-dependent apoptosis and cell cycle arrest. To counteract prolonged blockage of transcription, the cell removes the RNAPIIo-blocking DNA lesions by transcription-coupled repair (TC-NER), a specialized subpathway of nucleotide excision repair (NER). Exposure of mice to UVB light or chemicals has elucidated that TC-NER is a critical survival pathway protecting against acute toxic and long-term effects (cancer) of genotoxic exposure. Deficiency in TC-NER is associated with mutations in the CSA and CSB genes giving rise to the rare human disorder Cockayne syndrome (CS). Recent data suggest that CSA and CSB play differential roles in mammalian TC-NER: CSB as a repair coupling factor to attract NER proteins, chromatin remodellers and the CSA- E3-ubiquitin ligase complex to the stalled RNAPIIo. CSA is dispensable for attraction of NER proteins, yet in cooperation with CSB is required to recruit XAB2, the nucleosomal binding protein HMGN1 and TFIIS. The emerging picture of TC-NER is complex: repair of transcription-blocking lesions occurs without displacement of the DNA damage-stalled RNAPIIo, and requires at least two essential assembly factors (CSA and CSB), the core NER factors (except for XPC-RAD23B), and TC-NER specific factors. These and yet unidentified proteins will accomplish not only efficient repair of transcription-blocking lesions, but are also likely to contribute to DNA damage signalling events.
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PMID:Transcription-coupled nucleotide excision repair in mammalian cells: molecular mechanisms and biological effects. 1816 77

It is well accepted that for transcriptional silencing in budding yeast, the evolutionarily conserved lysine deacetylase Sir2, in concert with its partner proteins Sir3 and Sir4, establishes a chromatin structure that prevents RNA polymerase II (Pol II) transcription. However, the mechanism of repression remains controversial. Here, we show that the recruitment of Pol II, as well as that of the general initiation factors TBP and TFIIH, occurs unimpeded to the silent HMRa1 and HMLalpha1/HMLalpha2 mating promoters. This, together with the fact that Pol II is Ser5 phosphorylated, implies that SIR-mediated silencing is permissive to both preinitiation complex (PIC) assembly and transcription initiation. In contrast, the occupancy of factors critical to both mRNA capping and Pol II elongation, including Cet1, Abd1, Spt5, Paf1C, and TFIIS, is virtually abolished. In agreement with this, efficiency of silencing correlates not with a restriction in Pol II promoter occupancy but with a restriction in capping enzyme recruitment. These observations pinpoint the transition between polymerase initiation and elongation as the step targeted by Sir2 and indicate that transcriptional silencing is achieved through the differential accessibility of initiation and capping/elongation factors to chromatin. We compare Sir2-mediated transcriptional silencing to a second repression mechanism, mediated by Tup1. In contrast to Sir2, Tup1 prevents TBP, Pol II, and TFIIH recruitment to the HMLalpha1 promoter, thereby abrogating PIC formation.
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PMID:Sir2 silences gene transcription by targeting the transition between RNA polymerase II initiation and elongation. 1839 Oct 20


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