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)

Evidence is presented that more than 300 bp of spacer sequences downstream of the 28S RNA coding sequence are part of the mouse rDNA transcription unit. Studies in two cell-free transcription systems as well as analysis of cellular RNA indicate that RNA polymerase I does not terminate within the 334 bp 3' terminal spacer sequences contained in the rDNA clone used. Quantitative hybridization data, S1 mapping experiments and Northern analysis of nuclear RNA showed that the 14 kb pre-rRNA molecules hybridize with the same efficiency to both the 28S and the 3' NTS specific DNA probe. This indicates that the rRNA precursor contains both at the 5' and 3' end several hundreds bases of external transcribed spacer sequences which are eliminated in subsequent processing reactions.
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PMID:Spacer sequences downstream of the 28S RNA coding region are part of the mouse rDNA transcription unit. 258 62

Analysis of the termination of transcription by yeast RNA polymerase I (Pol I) using in vitro run-on experiments in both isolated nuclei and permeabilized cells demonstrated that Pol I does not traverse the whole intergenic spacer separating consecutive 37S operons, but terminates transcription before reaching the 5S rRNA gene, that is within NTS 1. In order to discriminate between processing and termination at the 3'-end generating sites previously identified in vivo in NTS 1 (T1, T2 and T3), fragments containing these sites were inserted into the middle of the reporter DNA of an artificial rRNA minigene. RNA isolated from yeast cells transformed with these minigenes was analyzed for the presence of transcripts derived from sequences both up- and downstream of the insert by Northern blot hybridization, reverse transcription analysis and S1 nuclease mapping. In accordance with previously obtained results T1 (+15 to +50) was found to behave as a processing site. T2 (+210) however was concluded to be an efficient, genuine Pol I terminator. In addition to T2, two other terminators were identified in NTS 1: T3A (at +690) and T3B (at +950). Surprisingly, when the 3' terminal part of NTS 2 was tested for its capacity to generate 3'-ends, another terminator (Tp) was found to be present at a position 300 bp upstream of the transcription initiation site of the 37S-rRNA operon.
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PMID:Termination of transcription by yeast RNA polymerase I. 268 55

Interactions between Escherichia coli RNA polymerase holoenzyme and three small plasmid DNAs (pSM1, pSM2, and pSM15) derived from the drug resistant factor R12 have been studied. These plasmids carry the copy number and incompatibility determinants, the origin of DNA replication and the rep gene(s) necessary for plasmid replication. They also contain the insertion element IS1 and the putative finO cistron. Thirteen DNA segments within the largest of the three plasmids (pSM2) were able to form either a binary and/or ternary complex with RNA polymerase. A unique strong binding site was mapped within the left end of IS1. Five binding sites were found within the rep-cop-inc region. Four of these are weak binding sites whereas the fifth does not form a stable binary complex and was detected by ternary complex formation. A strong binding site was located in the putative finO region whereas the remaining six binding sites are located in regions with unidentified genetic functions.
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PMID:Mapping of RNA polymerase binding sites in R12 derived plasmids carrying the replication-incompatibility region and the insertion element IS1. 629 71

Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the gray polyketide spore pigment, and such white (whi) mutants have been used to define 13 sporulation loci. whiN, one of five new whi loci identified in a recent screen of NTG (N-methyl-N'-nitro-N-nitrosoguanidine)-induced whi strains (N. J. Ryding et al., J. Bacteriol. 181:5419-5425, 1999), was defined by two mutants, R112 and R650. R650 produced frequent spores that were longer than those of the wild type. In contrast, R112 produced long, straight, undifferentiated hyphae, although rare spore chains were observed, sometimes showing highly irregular septum placement. Subcloning and sequencing showed that whiN encodes a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors and that the sigma factor has an unusual N-terminal extension of approximately 86 residues that is not present in other sigma factors. A constructed whiN null mutant failed to form aerial mycelium (the "bald" phenotype) and, as a consequence, whiN was renamed bldN. This observation was not totally unexpected because, on some media, the R112 point mutant produced substantially less aerial mycelium than its parent, M145. The bldN null mutant did not fit simply into the extracellular signaling cascade proposed for S. coelicolor bld mutants. Expression of bldN was analyzed during colony development in wild-type and aerial mycelium-deficient bld strains. bldN was transcribed from a single promoter, bldNp. bldN transcription was developmentally regulated, commencing approximately at the time of aerial mycelium formation, and depended on bldG and bldH, but not on bldA, bldB, bldC, bldF, bldK, or bldJ or on bldN itself. Transcription from the p1 promoter of the response-regulator gene bldM depended on bldN in vivo, and the bldMp1 promoter was shown to be a direct biochemical target for sigma(BldN) holoenzyme in vitro.
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PMID:sigma(BldN), an extracytoplasmic function RNA polymerase sigma factor required for aerial mycelium formation in Streptomyces coelicolor A3(2). 1091 95

The Balbiani ring 3 (BR3) gene contains 38 introns, and more than half of them are co-transcriptionally excised. We have determined the in situ structure of the active BR3 gene by electron tomography. Each of the 20-25 nascent transcripts on the gene is present together with splicing factors and the RNA polymerase II in a nascent transcript and splicing complex, here called the NTS complex. The results indicate that extensive changes in overall shape, substructure and molecular mass take place repeatedly within an NTS complex as it moves along the gene. The volume and calculated mass of the NTS complexes show that, maximally, one complete spliceosome is assembled on the multi-intron transcript at any given time point. The structural data show that the spliceosome is not a structurally well-defined unit in situ and that the C-terminal domain of the elongating RNA polymerase II cannot carry spliceosomal components for all introns in the BR3 transcript. Our data indicate that spliceosomal factors are continuously added to and released from the NTS complexes during transcription elongation.
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PMID:In situ transcription and splicing in the Balbiani ring 3 gene. 1135 Sep 46

1. Benzodiazepines (BZ) and barbiturates both potentiate chloride currents through GABA(A) receptors to enhance inhibition. However, unlike barbiturates BZ do not impair autonomic control of heart rate. We hypothesised that BZ might not significantly potentiate GABAergic transmission in the caudal nucleus of the solitary tract (cNTS), which is critically important for mediating the baroreceptor reflex. 2. In rat brain slices the BZ agonists chlordiazepoxide and midazolam (2 and 50 microM) did not significantly enhance currents evoked by GABA in voltage-clamped cNTS neurones. Chlordiazepoxide (50 microM) reversibly increased electrically evoked IPSPs in 5/10 rostral NTS (rNTS) neurones but only in 2/10 cNTS neurones. Pentobarbitone (50-100 microM) was effective in enhancing GABA(A)-mediated responses in all NTS neurones. An inverse BZ agonist, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM; 1 or 10 microM), failed to depress GABA-induced currents in the cNTS. 3. Microinjections of midazolam (10 and 100 microM solutions) into the cNTS did not affect the baroreceptor reflex (P > 0.2) while pentobarbitone (100 microM) significantly and reversibly depressed it (gain decrease to 53 +/- 11 % of control, P < 0.01). 4. Reverse transcriptase polymerase chain reaction revealed the presence of alpha(1), alpha(2), beta(2), beta(3) and gamma(2) GABA(A) receptor subunit mRNA in the cNTS. No alternatively spliced variants of the alpha(1)- and gamma(2)-subunits were revealed. Moreover, GABA(A) epsilon-unit mRNA was found in both the cNTS and rNTS as two alternatively spliced transcripts. 5. Immunocytochemical analysis revealed numerous GABA(A) epsilon-subunit-positive neurones within the cNTS with significantly fewer epsilon-subunit-positive cells in the rNTS. 6. As incorporation of the epsilon-subunit in recombinant GABA(A) receptors may confer BZ insensitivity we propose that the paucity of BZ actions in the cNTS is due to a high level of epsilon-subunit expression. This is the first demonstration of a possible physiological impact of the epsilon-subunit on native GABA(A) receptors.
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PMID:GABA(A) receptor epsilon-subunit may confer benzodiazepine insensitivity to the caudal aspect of the nucleus tractus solitarii of the rat. 1169 72

N protein of the Escherichia coli phage lambda (lambdaN) is involved in antitermination, a transcription regulatory process that is essential for the expression of delayed early genes during phage lytic development. lambdaN is an intrinsically unstructured protein that possesses three distinct binding sites interacting with the carboxy terminus of the E. coli host factor protein NusA, the viral nutBoxB-RNA, and RNA polymerase, respectively. Heteronuclear NMR experiments with lambdaN(1-53) in complex with NusA(339-495) revealed that upon complex formation the lambdaN-binding interface, lambdaN(34-47), adopts a rigid structure. NMR data also indicate the induction of a weak helical structure in the nutboxB RNA-binding region lambdaN(1-22) upon binding to NusA(339-495) even in the absence of RNA. Titration experiments of the lambdaN(1-53)-nutBoxB RNA complex with NusA(339-495) revealed that the ternary complex can be described in terms of two structurally independent binary interactions. Furthermore, chemical-shift perturbation experiments with different NusA constructs and different lambdaN peptides showed that only NusA(353-416) is involved in lambdaN binding. We found that only one molecule of NusA(339-426) binds to one molecule of lambdaN(1-53). We also clarified the role of the lambdaN-binding region and could show that N41-R47 also binds to NusA(339-495). Furthermore, we observe that lambdaN(1-22) adopts a helical fold upon binding to NusA(339-495), in agreement with one of the theoretical models of lambdaN action.
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PMID:Interaction of the intrinsically unstructured phage lambda N Protein with Escherichia coli NusA. 1658 89

Nitrate tolerance is associated with an enhanced superoxide anion (O(2)(-)) production and may be attenuated by statins as they interact with the two main endothelial NO synthase (eNOS) and NAD(P)H oxidase pathways involved in this oxidative stress. Groups of wild-type (wt, C57Bl/6J) and eNOS knock-out mice (eNOS(-/-)) received rosuvastatin (20 mg kg(-1) day(-1) p.o.) for 5 weeks and a cotreatment with the statin plus nitroglycerin (NTG; 30 mg kg(-1) day(-1), subcutaneous injections b.i.d.) for the last 4 days. Another group received only NTG (30 mg kg(-1) d(-1), b.i.d. for 4 days) and finally control mice from both strains received no treatment. Rings of thoracic aortas from these groups were studied in organ baths. Relaxations to NTG (0.1 nM-0.1 mM) were determined on thromboxane analogue (U44619)-precontracted rings and O(2)(-) production (RLU 5 s(-1) mg(-1) of total protein content) was assessed in aorta homogenates with the lucigenin-enhanced chemiluminescence technique. Reverse transcriptase-polymerase chain reaction analysis was performed on aortas from both mice strains. In vivo NTG treatment induced a significant rightward shift of the concentration-effect curve to NTG compared to control group. There was, however, no cross-tolerance with non-nitrate sources of NO (unaltered response to acetylcholine in wt group). The rosuvastatin + NTG cotreatment was able to protect against the development of nitrate tolerance in both mice strains and L-mevalonate abolished this protective effect of rosuvastatin. In vivo treatment with apocynin, a purported NAD(P)H oxidase inhibitor, also produced a similar protection to that observed with rosuvastatin in both strains. Superoxide anion formation was increased after NTG treatment in both mice strains and the rosuvastatin + NTG cotreatment was able to reduce that production. Moreover, rosuvastatin treatment abolished the increase in gp91phox mRNA (an endothelial membrane NAD(P)H oxidase subunit) expression induced by in vivo exposure to NTG. These findings suggest that long-term rosuvastatin treatment protects against nitrate tolerance by counteracting NTG-induced increase in O(2)(-) production, probably via a direct interaction with the NAD(P)H oxidase pathway.
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PMID:Rosuvastatin treatment protects against nitrate-induced oxidative stress in eNOS knockout mice: implication of the NAD(P)H oxidase pathway. 1663 68

The rDNA cluster is the genetic locus encoding the ribosomal RNAs and physically defines where ribosomes begin to be assembled. In the yeast Saccharomyces cerevisiae, the highly repetitive structure of this locus makes it a very interesting target for studies about genome stability, chromatin-mediated transcriptional silencing and progression of aging. In fact, recombination among the repeated units is suppressed in a WT cell. Moreover, when genes transcribed by RNA polymerase II are inserted in the rDNA cluster, their transcription is silenced. Finally, the formation of rDNA minicircles (ERCs) has been shown to be one of the causes of aging in yeast. DNA topoisomerase I have been shown to suppress recombination specifically at the rDNA of S.cerevisiae. Moreover, also the chromatin structure of this locus is affected in a top1 strain, because rDNA specific transcriptional silencing is abolished. Nonetheless, the molecular basis of how this enzyme interferes with these functions is yet unknown. Here are reported results obtained by in vivo studies of DNA protein interactions occurring on the rDNA locus. The analyses include a fine mapping of nucleosome positioning; RNA polymerase I transcription factors and DNA topoisomerase I cleavage sites. Important conclusions can be drawn: i) nucleosome positioning in the Non Transcribed Spacer is not affected by RNA polymerase I transcription; ii) the RNA polymerase I transcription factors bind DNA in vivo with a defined hierarchy; iii) the DNA topoisomerase I cleaves the NTS in very specific sites, but cleavage is not induced by RNA polymerase I transcription. These in vivo studies help to characterize the molecular basis of important phenomena as the transcriptional silencing and genome stability in yeast.
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PMID:DNA protein interactions at the rRNA of Saccharomyces cerevisiae. 1772 48

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) (1-3). Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems (4). The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster (5). The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs (6-8). These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence (9). A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity(10). Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA (11,12) . These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases (4). Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.
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PMID:Substrate generation for endonucleases of CRISPR/cas systems. 2298 8


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