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)

The regulation of gene expression depends critically upon chromatin structure. Transcription of protein-coding genes can be reconstituted on naked DNA with only the general transcription factors and RNA polymerase II. This minimal system cannot transcribe DNA packaged into chromatin, indicating that accessory factors may facilitate access to DNA. Two classes of accessory factor, ATP-dependent chromatin-remodelling enzymes and histone acetyltransferases, facilitate transcription initiation from chromatin templates. FACT (for facilitates chromatin transcription) is a chromatin-specific elongation factor required for transcription of chromatin templates in vitro. Here we show that FACT comprises a new human homologue of the Saccharomyces cerevisiae Spt16/Cdc68 protein and the high-mobility group-1-like protein structure-specific recognition protein-1. Yeast SPT16/CDC68 is an essential gene that has been implicated in transcription and cell-cycle regulation. Consistent with our biochemical analysis of FACT, we provide evidence that Spt16/Cdc68 is involved in transcript elongation in vivo. Moreover, FACT specifically interacts with nucleosomes and histone H2A/H2B dimers, indicating that it may work by promoting nucleosome disassembly upon transcription. In support of this model, we show that FACT activity is abrogated by covalently crosslinking nucleosomal histones.
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PMID:The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins. 1042 73

To investigate the in vitro transcription by bacteriophage T7 RNA polymerase of oligonucleosomes lacking histone H2A x H2B dimers, templates were assembled from histone (H3 x H4)(2) tetramers with and without the complementary amount of H2A x H2B dimers and two different DNA species: pGEMEX-1, devoid of nucleosome positioning sequences, and T7-207-18, which contains downstream from the promoter 18 tandem repeats of a 207-bp positioning sequence. Assembly with core histone octamers affects pGEMEX-1 transcription mainly at the initiation level, while T7-207-18 is almost exclusively inhibited at the level of elongation. With both DNA templates and under different salt conditions, RNA synthesis is much more efficient on oligonucleosomes containing only (H3 x H4)(2) tetramers than on those with whole histone octamers. Under conditions promoting a low transcription rate, it is unambiguously shown with pGEMEX-1 that the block to initiation due to the presence of core histone octamers is substantially removed when (H3 x H4)(2) is substituted for the whole octamer. With T7-207-18, under assay conditions allowing transcription of the whole coding region of the naked DNA, analysis of the transcription products indicates that RNA elongation on the template containing (H3 x H4)(2) tetramers takes place as easily as on free DNA, in contrast with the significant inhibition observed in the presence of whole histone octamers.
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PMID:Transcription of DNA templates associated with histone (H3 x H4)(2) tetramers. 1051 Feb 81

Several factors were evaluated to determine their role in facilitating the presence of transcription-induced stresses in a circular DNA. Transcription was done with T7 RNA polymerase in the presence of E. coli topoisomerase I and closed circular DNA. Positive stress was observed in hypotonic conditions or when one of the polyamines, spermidine or spermine, were present. Polycations such as polylysine, polyarginine, histone H1, histones H2A-H2B, and protamine were observed to induce minimal positive stress. It is known that polyamines influence DNA structure by causing both self-association and sequence-specific structural alterations (polyamine-induced localized bending). Experimental evidence indicates that the likely cause of the positive stress is the induced bending. In order to evaluate protein-mediated bending, transcription was done on nucleosomes. A minimum of three nucleosomes on a DNA of 6055 bp was sufficient to generate very high levels of positive stress. Histones H3-H4 in the absence of H2A-H2B were responsible for this effect. Since these histones by themselves are able to maintain negative coils on DNA, it is concluded that protein-mediated bending is yet another mechanism for placing rotational restriction on DNA. The bending of DNA by either polyamines or histones is an effective mechanism for promoting transcription-induced stresses at physiological ionic strength.
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PMID:In vitro studies on the maintenance of transcription-induced stress by histones and polyamines. 1061 64

DNA packaging into chromatin presents a strong barrier to RNA polymerase II transcription. In this issue of Molecular Cell, Kireeva et al. describe a minimal system to examine polymerase II transcription through a positioned nucleosome and show, surprisingly, that transcription leads to the displacement of an H2A.H2B dimer from the nucleosome without altering nucleosome position.
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PMID:How does Pol II overcome the nucleosome barrier? 1193 62

RNA polymerase II (Pol II) must transcribe genes in a chromatin environment in vivo. We examined transcription by Pol II through nucleosome cores in vitro. At physiological and lower ionic strengths, a mononucleosome imposes a strong block to elongation, which is relieved at increased ionic strength. Passage of Pol II causes a quantitative loss of one H2A/H2B dimer but does not alter the location of the nucleosome. In contrast, bacteriophage SP6 RNA polymerase (RNAP) efficiently transcribes through the same nucleosome under physiological conditions, and the histone octamer is transferred behind SP6 RNAP. Thus, the mechanisms for transcription through the nucleosome by Pol II and SP6 RNAP are clearly different. Moreover, Pol II leaves behind an imprint of disrupted chromatin structure.
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PMID:Nucleosome remodeling induced by RNA polymerase II: loss of the H2A/H2B dimer during transcription. 1193 51

The passage of RNA polymerase II across eukaryotic genes is impeded by the nucleosome, an octamer of histones H2A, H2B, H3 and H4 dimers. More than a dozen factors in the yeast Saccharomyces cerevisiae are known to facilitate transcription elongation through chromatin. In order to better understand the evolution and function of these factors, their sequences have been compared with known protein, EST and DNA sequences. Elongator subcomplex components Elp4p and Elp6p are shown to be homologues of ATPases, yet with substitutions of amino acids critical for ATP hydrolysis, and novel orthologues of Elp5p are detectable in human, and other animal, sequences. The yeast CP complex is shown to contain a likely inactive homologue of M24 family metalloproteases in Spt16p/Cdc68p and a 2-fold repeat in Pob3p, the orthologue of mammalian SSRP1. Archaeal DNA-directed RNA polymerase subunit E" is shown to be the orthologue of eukaryotic Spt4p, and Spt5p and prokaryotic NusG are shown to contain a novel 'NGN' domain. Spt6p is found to contain a domain homologous to the YqgF family of RNases, although this domain may also lack catalytic activity. These findings imply that much of the transcription elongation machinery of eukaryotes has been acquired subsequent to their divergence from prokaryotes.
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PMID:Novel domains and orthologues of eukaryotic transcription elongation factors. 1220 48

We have previously shown that nucleosomes act as a strong barrier to yeast RNA polymerase II (Pol II) in vitro and that transcription through the nucleosome results in the loss of an H2A/H2B dimer. Here, we demonstrate that Escherichia coli RNA polymerase (RNAP), which never encounters chromatin in vivo, behaves similarly to Pol II in all aspects of transcription through the nucleosome in vitro. The nucleosome-specific pausing pattern of RNAP is comparable with that of Pol II. At physiological ionic strength or lower, the nucleosome blocks RNAP progression along the template, but this barrier can be relieved at higher ionic strength. Transcription through the nucleosome by RNAP results in the loss of an H2A/H2B dimer, and the histones that remain in the hexasome retain their original positions on the DNA. The results were similar for elongation complexes that were assembled from components (oligonucleotides and RNAP) and elongation complexes obtained by initiation from the promoter. The data suggest that eukaryotic Pol II and E. coli RNAP utilize very similar mechanisms for transcription through the nucleosome. Thus, bacterial RNAP can be used as a suitable model system to study general aspects of chromatin transcription by Pol II. Furthermore, the data argue that the general elongation properties of polymerases may determine the mechanism used for transcription through the nucleosome.
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PMID:Bacterial polymerase and yeast polymerase II use similar mechanisms for transcription through nucleosomes. 1285 91

The FACT (facilitates chromatin transcription) complex is required for transcript elongation through nucleosomes by RNA polymerase II (Pol II) in vitro. Here, we show that FACT facilitates Pol II-driven transcription by destabilizing nucleosomal structure so that one histone H2A-H2B dimer is removed during enzyme passage. We also demonstrate that FACT possesses intrinsic histone chaperone activity and can deposit core histones onto DNA. Importantly, FACT activity requires both of its constituent subunits and is dependent on the highly acidic C terminus of its larger subunit, Spt16. These findings define the mechanism by which Pol II can transcribe through chromatin without disrupting its epigenetic status.
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PMID:FACT facilitates transcription-dependent nucleosome alteration. 1293 97

Transcription through a multinucleosomal template was studied to determine why histones are released to the nascent RNA. It was first determined in competition experiments between DNA and RNA that histones H2A and H2B have a 20-fold preference for binding RNA over DNA; a preference was not seen for histones H3 and H4. Histones H3 and H4 would preferentially bind RNA, provided they were in an octameric complex with H2A and H2B. In transcription studies with T7 RNA polymerase, H3 and H4 were transferred to the nascent RNA, provided the template was linear. If the DNA was topologically restrained, which is a condition that more closely maintains transcription-induced stresses, H3 and H4 would not release. Histones H3 and H4 would be released from this template when H2A and H2B were present, a release that was enhanced by the presence of nucleosome assembly protein-1 (NAP1). Since a small quantity of H2A and H2B is sufficient to facilitate this transfer, it is proposed that H2A and H2B function to repeatedly shuttle H3 and H4 from the template DNA to the RNA. Cross-linked histones (dimethylsuberimidate-cross-linked octamer) were reconstituted into nucleosomes and found to be transferred to the RNA at the same frequency as un-cross-linked histones, an indication that such large complexes can be released during transcription. Transcription was carried out in the presence of Escherichia coli topoisomerase I so that positive coils would accumulate on the DNA. Histones H3 and H4 would again not be transferred from this DNA, unless H2A and H2B were present. In this instance, however, when NAP1 was present, the shuttling of H3 and H4 to the RNA caused a significant depletion of H2A and H2B from the positively coiled DNA. These results are discussed with regard to current models for transcription through nucleosomes.
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PMID:Histone release during transcription: NAP1 forms a complex with H2A and H2B and facilitates a topologically dependent release of H3 and H4 from the nucleosome. 1499 73

Chromatin packages DNA tightly into the eukaryotic nucleus and maintains its proper functioning. Recent studies suggest the existence of two distinct mechanisms of progression of RNA polymerases through chromatin. The first is characteristic of eukaryotic RNA polymerase III, bacteriophage RNA polymerases, and probably ATP-dependent chromatin remodeling complexes. In this mechanism, nucleosomes are translocated without release of the octamer into solution. By contrast, transcription by RNA polymerase II (Pol II) involves displacement of one H2A-H2B dimer. Nucleosomes can present a barrier for transcribing Pol II that can be regulated in vivo. Analysis of the mechanisms of transcription through chromatin should provide important information about mechanisms of chromatin remodeling and gene regulation at the level of transcript elongation.
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PMID:Chromatin remodeling by RNA polymerases. 1500 70


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