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 diverse functions of Saccharomyces cerevisiae RNA polymerase II are partitioned among its 12 subunits, designated RPB1-RPB12. Although multiple functions have been assigned to the three largest subunits, RPB1, RPB2, and RPB3, the functions of the remaining smaller subunits are unknown. We have determined the function of one of the smaller subunits, RPB9, by demonstrating that it is necessary for accurate start site selection. Transcription in the absence of RPB9 initiates farther upstream at new and previously minor start sites both at the CYC1 promoter in vitro and at the CYC1, ADH1, HIS4, H2B-1, and RPB6 promoters in vivo. Immunoprecipitation of RNA polymerase II from cells lacking the RPB9 gene revealed that all of the remaining 11 subunits are assembled into the enzyme, suggesting that the start site defect is attributable solely to the absence of RPB9. In support of this hypothesis, we have shown that addition of wild-type recombinant RPB9 completely corrects for the start site defect seen in vitro. A mutated recombinant RPB9 protein, with an alteration in a metal-binding domain required for high temperature growth and accurate start site selection in vivo, was at least 10-fold less effective at correcting the start site defect in vitro. RPB9 appears to play a unique role in transcription initiation, as the defects revealed in its absence are distinct from those seen with mutants in RNA polymerase subunit RPB1 and factor e (TFIIB), two other yeast proteins also involved in start site selection.
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PMID:RNA polymerase II subunit RPB9 is required for accurate start site selection. 788 69

Histone octamers or histone H3/H4 tetramers were reconstituted onto either closed circular plasmids containing a single Xenopus 5S rRNA gene or a reiterated array of Lytechinus 5S rRNA genes. All "reconstitutes" were found to undergo both Na(+)-dependent and Mg(2+)-dependent compaction. However, in each case, the compaction of nucleosomal templates containing H2A/H2B was much more extensive than compaction of templates containing only H3/H4 tetramers. Inclusion of 5 mM MgCl2 in the transcription buffer increased the level of compaction of nucleosomal templates and led to a marked inhibition of both transcription initiation and elongation by RNA polymerase III. The inhibitory effect of Mg2+ was reduced significantly when DNA templates contained only H3/H4 tetramers, consistent with their lesser extent of Mg(2+)-dependent compaction. Thus, the removal of histones H2A/H2B from nucleosomal arrays enhances gene activity, in part because of decreased levels of chromatin folding.
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PMID:A role for histones H2A/H2B in chromatin folding and transcriptional repression. 813 97

The interactions of histones H2A,H2B and H3,H4 with closed circular DNA maintained in either a positively or negatively coiled state have been studied. The interactions were assayed by measuring the rate at which negative stress was stored in the DNA by the histones and by the salt concentration sufficient to cause dissociation on sucrose gradients. Additional experiments were performed in which DNAs of substantially different molecular weights and opposite topological states were mixed with the histones in order to study histone mobility under varied conditions. This mobility was characterized by separating the complexes on sucrose gradients and by analyzing the DNA's topological state after topoisomerase I treatment. Histones H3,H4 were found to differ substantially from histones H2A,H2B with regard to the DNA topology with which they prefer to interact. The results are consistent with a model in which transcription-induced positive stress in advance of the RNA polymerase unfolds the nucleosome to facilitate the release of H2A,H2B. The data are also consistent with a model in which histones H3,H4 remain associated with the DNA during polymerase passage and serve as a nucleation site for the reassociation of H2A,H2B. The rapid production of transcription-induced negative stress in the wake of a polymerase would have substantial importance in facilitating the reassociation of histones H2A,H2B.
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PMID:Dynamics of the interactions of histones H2A,H2B and H3,H4 with torsionally stressed DNA. 818 Jan 62

A histone-DNA transcription template has been assembled, by dialysis against decreasing salt concentrations, from pGEMEX-1 (4 kilobases), a plasmid containing a promoter for bacteriophage T7 RNA polymerase, and from isolated histone (H3.H4)2 tetramers. Electron microscopy after psoralen cross-linking shows that each histone tetramer protects approximately 80 base pairs of DNA from psoralen action and that, under the employed conditions, an average of 15 tetramer particles are assembled per DNA molecule. This (H3.H4)2-DNA template is efficiently transcribed in vitro by T7 RNA polymerase as compared to naked DNA. The presence of (H3.H4)2 tetramers does not affect initiation, in contrast with the complete histone octamer, (H2A.H2B.H3.H4)2, assembled with the complementary addition of H2A.H2B dimers, which causes transcriptional inhibition mainly by blocking initiation.
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PMID:Efficient transcription of a DNA template associated with histone (H3.H4)2 tetramers. 825 98

Negative co-factor 2 (NC2) regulates transcription of the class II genes through binding to TFIID and inhibition of pre-initiation complex formation. We have isolated and cloned NC2, and investigated the molecular mechanism underlying repression of transcription. NC2 consists of two subunits, termed NC2alpha and NC2beta, the latter of which is identical to Dr1. The NC2 subunits dimerize and bind to TATA binding protein (TBP)-promoter complexes via histone fold domains of the H2A-H2B type. Repression of basal transcription requires the histone fold and carboxy-terminal domains of the NC2 subunits. Several mechanisms probably contribute to transcriptional repression. Binding of NC2 inhibits association of TFIIB with TBP-promoter complexes. NC2 binds directly to DNA, and binding of NC2 to TBP-promoter complexes affects the conformation of DNA, which could be one cause for the inhibition of TFIIB. In addition, multimerization of repressor-TBP complexes on DNA might inhibit the assembly of the pre-initiation complex. We suggest that binding of the repressor to TRP-promoter complexes establishes a mechanism that controls the rate of transcription by RNA polymerase II.
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PMID:A mechanism for repression of class II gene transcription through specific binding of NC2 to TBP-promoter complexes via heterodimeric histone fold domains. 867 Aug 11

Chlamydia trachomatis is an obligate intracellular pathogen, long recognized as an agent of blinding eye disease and more recently as a common sexually transmitted infection. Recently, two eukaryotic histone H1-like proteins, designated Hc1 and Hc2, have been identified in Chlamydia. Expression of Hc1 in recombinant Escherichia coli produces chromatin condensation similar to nucleoid condensation observed late in the parasite's own life cycle. In contrast, chromatin decondensation, observed during the early life cycle, accompanies down-regulation and nondetection of Hc1 and Hc2 among internalized organisms. We reasoned that the early upstream open reading frame (EUO) gene product might play a role in Hc1 degradation and nucleoid decondensation since it is expressed very early in the chlamydial life cycle. To explore this possibility, we fused the EUO coding region between amino acids 4 and 177 from C. trachomatis serovar Lz with glutathione S-transferase (GST) and examined the effects of fusion protein on Hc1 in vitro. The purified fusion protein was able to digest Hc1 completely within 1 h at 37 degrees C. However, GST alone exhibited no Hc1-specific proteolytic activity. The chlamydial EUO-GST gene product also cleaves very-lysine-rich calf thymus histone H1 and chicken erythrocyte histone H5 but displays no measurable activity towards core histones H2A, H2B, H3, and H4 or chlamydial RNA polymerase alpha-subunit. This proteolytic activity appears sensitive to the serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) and aspartic protease inhibitor pepstatin but resistant to high temperature and other broad-spectrum protease inhibitors. The proteolytic activity specified by the EUO-GST fusion product selectively digested the C-terminal portion of chlamydial Hc1, the domain involved in DNA binding, while leaving the N terminus intact. At a molar equivalent ratio of 1:1 between Hc1 and DNA, the EUO gene product cleaves Hc1 complexed to DNA and this cleavage appears sufficient to initiate dissociation of DNA-Hc1 complexes. However, at a higher molar equivalent ratio of Hc1/DNA (10:1), there is partial protection conferred upon Hc1 to an extent that prevents dissociation of DNA-Hc1 complexes.
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PMID:The chlamydial EUO gene encodes a histone H1-specific protease. 929 54

Upstream binding factor (UBF) is a vertebrate RNA polymerase I transcription factor that can bend and wrap DNA. To investigate UBF's likely role as an architectural protein of rRNA genes organized in chromatin, we tested UBF's ability to bind rRNA gene enhancers assembled into nucleosome cores (DNA plus core histones) and nucleosomes (DNA plus core histones plus histone H1). UBF bound with low affinity to nucleosome cores formed with enhancer DNA probes of 162 bp. However, on nucleosome cores which contained approximately 60 bp of additional linker DNA, UBF bound with high affinity similar to its binding to naked DNA, forming a ternary DNA-core histone-UBF complex. UBF could be stripped from ternary complexes with competitor DNA to liberate nucleosome cores, rather than free DNA, suggesting that UBF binding to nucleosome cores does not displace the core histones H2A, H2B, H3, and H4. DNase I, micrococcal nuclease, and exonuclease III footprinting suggests that UBF and histone H1 interact with DNA on both sides flanking the histone octamer. Footprinting shows that UBF outcompetes histone H1 for binding to a nucleosome core and will displace, if not dissociate, H1 from its binding site on a preassembled nucleosome. These data suggest that UBF may act to prevent or reverse the assembly of transcriptionally inactive chromatin structures catalyzed by linker histone binding.
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PMID:Nucleosome binding by the polymerase I transactivator upstream binding factor displaces linker histone H1. 931 41

Sin mutations in Saccharomyces cerevisiae alleviate transcriptional defects that result from the inactivation of the yeast SWVI/SNF complex. We have investigated the structural and functional consequences for the nucleosome of Sin mutations in histone H3. We directly test the hypothesis that mutations in histone H3 leading to a SWI/SNF-independent (Sin) phenotype in yeast lead to nucleosomal destabilization. In certain instances this is shown to be true; however, nucleosomal destabilization does not always occur. Topoisomerase I-mediated relaxation of minichromosomes assembled with either mutant histone H3 or wild-type H3 together with histones H2A, H2B, and H4 indicates that DNA is constrained into nucleosomal structures containing either mutant or wild-type proteins. However, nucleosomes containing particular mutant H3 molecules (R116-H and T118-I) are more accessible to digestion by micrococcal nuclease and do not constrain DNA in a precise rotational position, as revealed by digestion with DNase I. This result establishes that Sin mutations in histone H3 located close to the dyad axis can destabilize histone-DNA contacts at the periphery of the nucleosome core. Other nucleosomes containing a distinct mutant H3 molecule (E105-K) associated with a Sin phenotype show very little change in nucleosome structure and stability compared to wild-type nucleosomes. Both mutant and wild-type nucleosomes continue to restrict the binding of either TATA-binding protein/transcription factor IIA (TFIIA) or the RNA polymerase III transcription machinery. Thus, different Sin mutations in histone H3 alter the stability of histone-DNA interactions to various extents in the nucleosome while maintaining the fundamental architecture of the nucleosome and contributing to a common Sin phenotype.
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PMID:Sin mutations of histone H3: influence on nucleosome core structure and function. 937 28

RNA polymerase I (Pol I) transcription in the yeast Saccharomyces cerevisiae is greatly stimulated in vivo and in vitro by the multiprotein complex, upstream activation factor (UAF). UAF binds tightly to the upstream element of the rDNA promoter, such that once bound (in vitro), UAF does not readily exchange onto a competing template. Of the polypeptides previously identified in purified UAF, three are encoded by genes required for Pol I transcription in vivo: RRN5, RRN9, and RRN10. Two others, p30 and p18, have remained uncharacterized. We report here that the N-terminal amino acid sequence, its mobility in gel electrophoresis, and the immunoreactivity of p18 shows that it is histone H3. In addition, histone H4 was found in UAF, and myc-tagged histone H4 could be used to affinity-purify UAF. Histones H2A and H2B were not detectable in UAF. These results suggest that histones H3 and H4 probably account for the strong binding of UAF to DNA and may offer a means by which general nuclear regulatory signals could be transmitted to Pol I.
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PMID:Histones H3 and H4 are components of upstream activation factor required for the high-level transcription of yeast rDNA by RNA polymerase I. 939 Oct 47

Histone-DNA templates for bacteriophage T7 RNA polymerase were assembled from a plasmid containing a promoter and a terminator for this polymerase, (H3 x H4)2 tetramers deprived of their tail domains, and H2A x H2B dimers. Histone (H3 x H4)2 tetramers lacking their terminal domains were obtained from trypsin-digested nucleosomal cores. The oligonucleosomal templates containing (H3 x H4)2 tetramers lacking their tail domains, like the control templates with intact core histone octamers, protect approximately 146 base pairs of DNA against micrococcal nuclease digestion. The transcriptional inhibition caused by the association of DNA with core histone octamers is significantly reduced upon elimination of the tail domains of the (H3 x H4)2 tetramers. Apparently, the terminal domains of (H3 x H4)2 must be present to block transcription efficiently. These results show the important inhibitory role played by the tail domains of the histone (H3 x H4)2 tetramers, suggesting the involvement of these regions in transcriptional regulation.
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PMID:Transcriptional inhibitory role of the tail domains of histone (H3 x H4)2 tetramers. 975 Jan 70


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