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

---

Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

T7 RNA polymerase selectively transcribes T7 genes during infection but is also involved in DNA replication, maturation and packaging. T7 lysozyme is an amidase that cuts a bond in the peptidoglycan layer of the cell wall, but it also binds T7 RNA polymerase and inhibits transcription, and it stimulates replication and packaging of T7 DNA. To better understand the roles of these two proteins during T7 infection, mutants of each were constructed or selected and their biochemical and physiological behavior analyzed. The amidase activity of lysozyme is needed for abrupt lysis and release of phage particles but appears to have no role in replication and packaging. The interaction between polymerase and lysozyme stimulates both replication and packaging. Polymerase mutants that gain the ability to grow normally in the absence of an interaction with lysozyme still fail to shut down late transcription and, remarkably, have become hypersensitive to inhibition when lysozyme is able to bind. These lysozyme-hypersensitive polymerases behave without lysozyme similarly to wild-type polymerase with lysozyme: both remain longer at the promoter before establishing a lysozyme-resistant elongation complex and both increase the length of pausing when elongation complexes encounter an eight-base recognition sequence involved in DNA packaging. Replication origins contain T7 promoters, but the role of T7 RNA polymerase in initiating replication is not understood well enough to more than speculate how the lysozyme-polymerase interaction stimulates replication. Maturation and packaging is apparently initiated through interaction between prohead-terminase complexes and transcription elongation complexes paused at the sequence TATCTGT(T/A), well conserved at the right-end of the concatemer junction of T7-like phages. A model that is consistent with the structure of an elongation complex and a large body of mutational and biochemical data is proposed to explain sequence-specific pausing and potential termination at the consensus recognition sequence (C/T)ATCTGT(T/A).
...
PMID:Multiple roles of T7 RNA polymerase and T7 lysozyme during bacteriophage T7 infection. 1522 15

Transcription initiation begins with recruitment of an RNA polymerase to a promoter. Polymerase-promoter interactions are retained until the nascent RNA is extended to 8-12 nucleotides. It has been proposed that accumulation of "strain" in the transcription complex and RNA displacement of promoter-polymerase interactions contribute to releasing the polymerase from the promoter, and it has been further speculated that too strong a promoter interaction can inhibit the release step, whereas a weak interaction may facilitate release. We examined the effects of partial deletion of the nontemplate strand on release of T7 RNA polymerase from the T7 promoter. T7 polymerase will initiate from such partially single-stranded promoters but binds them with higher affinity than duplex promoters. We found that release on partially single-stranded promoters is strongly inhibited. The inhibition of release is not due to an indirect effect on transcription complex structure or loss of specific polymerase-nontemplate strand interactions, because release on partially single-stranded templates is recovered if the interaction with the promoter is weakened by a promoter base substitution. This same substitution also appears to allow the polymerase to escape more readily from a duplex promoter. Our results further suggest that template-nontemplate strand reannealing drives dissociation of abortive transcripts during initial transcription and that loss of interactions with either the nontemplate strand or duplex DNA downstream of the RNA lead to increased transcription complex slippage during initiation.
...
PMID:Multiple roles for the T7 promoter nontemplate strand during transcription initiation and polymerase release. 1556 15

The increasing incidence of tuberculosis and other mycobacterial infections due to AIDS epidemic resulted in the need of rapid and accurate identification of isolated mycobacteria. The correct identification result leads to the selection of an appropriate therapeutic regimen. Polymerase chain reaction and restriction enzyme analysis (PCR-REA) has been developed since 1992 and used as the rapid method for identifying mycobacteria. Several genes or sequences have been used as an amplified target for PCR-REA. The present study aims to evaluate the potential use of PCR-REA of gene-encoding heat shock protein 65 kDa (hsp65) and beta-subunit RNA polymerase (rpoB) for the identification of mycobacteria compared with conventional biochemical identification. Two hundreds clinical isolates, consisting of 50 isolates of Mycobacterium tuberculosis and 150 isolates of nontuberculous mycobacteria (NTM), were submitted for identification using PCR-REA and biochemical method. The results demonstrated that PCR-REA identified 188 isolates of both M. tuberculosis and NTM concordantly with biochemical identification. Discordant identification results obtained from 12 isolates, comprised of 8 M. scrofulaceum, 1 M. avium complex, 1 M. malmoense, 1 M. terrae complex, and 1 M. chelonae/abscessus. Overall, the concordant percentage of results obtained from PCR-REA compared with biochemical method was 100%, 98.8%, and 83.3% for M. tuberculosis complex, rapidly growing, and slowly growing mycobacteria, respectively, and the results of hsp65 PCR-REA was in agreement with those obtained from rpoB PCR-REA. From this study, PCR-REA appears to be a simple, rapid, and reliable method for identifying mycobacteria in a routine microbiology laboratory.
...
PMID:Comparative evaluation of polymerase chain reaction and restriction enzyme analysis: two amplified targets, hsp65 and rpoB, for identification of cultured mycobacteria. 1576 1

Eukaryotic chromosomal DNA is densely packaged in the nucleus and organized into discrete domains of active and inactive chromatin. Gene loci that are activated during the process of cell differentiation undergo changes that result in modifications of specific histone tail residues and in loosening of chromatin structure. The beta-globin genes are expressed exclusively in erythroid cells. High-level expression of these genes is mediated by a locus control region (LCR), a powerful DNA regulatory element composed of several DNase I hypersensitive (HS) sites and located far upstream of the beta-globin genes. Here we show that RNA polymerase II and specific histone modifications that mark transcriptionally active chromatin domains are associated with the LCR core elements HS2 and HS3 in murine embryonic stem cells prior to differentiation along the erythroid lineage. At this stage HS3 is abundantly transcribed. After in vitro differentiation, RNA Polymerase II can also be detected at the embryonic epsilon- and adult beta-globin genes. These results are consistent with the hypothesis that activation of the beta-globin gene locus is initiated by protein complexes recruited to the LCR.
...
PMID:Recruitment of transcription complexes to the beta-globin locus control region and transcription of hypersensitive site 3 prior to erythroid differentiation of murine embryonic stem cells. 1644 61

Arom gene, encoding a single polypeptide that catalyses steps two to six of the aromatic amino acid (phenylalanine, tyrosine and tryptophan) biosynthetic pathway, has been amplified from Scleortinia sclerotiorum genomic DNA by PCR and sequenced. In order to identify the fragment encoding AROM protein experimentally and search a method of obtaining the enzyme in a large amount, the open reading frame of arom gene of S. sclerotiorum was amplified by Pyrobest DNA Polymerase and inserted between Kpn I and Not I sites of the vector pYES2 to construct the expression vector pYES2-arom. The construct was transformed into Saccharomyces cerevisiae H158 by the method of LiAc/ SSDNA/PEG. The rate of transformation was 2 x 10(2)/microg DNA, which was enough for the selection of the positive transformants. PCR using the extracted plasmids as the templates and restriction enzyme analysis of the plasmids extracted from E. coli cells transformed by the above plasmids were performed respectively to screen the positive S. cerevisiae transformants since the copy number of the plasmid in S. cerevisiae was low. Subsequently, the transformant activated by the SC-U medium containing 2% raffinose was inoculated into the SC-U medium containing 2% galactose and the SC-U medium containing 2% glucose respectively to induce and depress the expression of the foreign arom gene. The results of RT-PCR analysis showed: there was not any DNA band in the negative control without the anti-transcriptase, which indicated there was no DNA contamination in the extracted total RNA; there was an expected DNA band in the positive control using the expression vector pYES2-arom as the template, which indicated the used amplification condition was proper; there was not any DNA band in the negative control using total RNA from the depressed transformant as the template, which indicated the DNA bands amplified from total RNA of the induced transformant were not false; there were the expected DNA bands in the samples using total RNA of the transformant induced for 48h, 60h, 72h or 84h as the templates, which indicated the heterogeneous arom gene was transcribed in S. cerevisiae H158 cells. The result of Northern hybridization was consistent with that of RT-PCR, and showed that arom gene of S. sclerotiorum had been transcribed in S. cerevisiae H158 cells when the cells were induced for 48h in the SC-U medium containing 2% galactose at 30 degrees C at 180r/min. 5-enolpyruvylshikimate-3-phosphate synthase activity of the transformant, which was one of AROM protein activities, was measured by estimating the rate of Pi release to check the expressed AROM protein was active or not. The results of enzyme assay in the different culture period indicated that the transformant had 5-enolpyruvylshikimate-3-phosphate synthase activity and the activity reached the peak when the transformant was induced for 72h in SC-U medium at 30 degrees C at 180r/min. The molecular weight of AROM protein is high, it exists in cytoplasm as a dimmer and its expression is controlled by the amounts of amino acids. Therefore, it is very difficult to purify the enzyme. A great lot protein can be obtained by heterogeneous expression. S. cerevisiae expression system has the merits of safe status, authentic posttranslational modification, fast cultivation etc. and usually is the first choice eukaryotic expression system. S. cerevisiae expression system of AROM protein from S. sclerotiorum was successfully constructed for the first time, which provided the basis for the research on the catalysis mechanism of the enzyme and an economical means of simultaneously synthesizing five aromatic amino acid biosynthetic pathway enzymes.
...
PMID:[Cloning and expression of arom gene of Sclerotinia sclerotiorum]. 1657 63

An investigation has been made of the RNA synthesized by chromatin-bound RNA polymerase from soybean hypocotyls (Glycine max var. Wayne). Polymerase activity is 4- to 5-fold higher with chromatin from tissue treated with 2,4-dichlorophenoxyacetic acid, a synthetic auxin, compared to untreated tissue. Thin layer chromatography of the RNA hydrolysis products and acrylamide gel electrophoresis of the RNA synthesized by the chromatin show that increased activity induced by 2,4-dichlorophenoxyacetic acid is due primarily to the production of longer RNA chains, with only 20 to 50% increase in the number of RNA chains. The observation that 2,4-dichlorophenoxyacetic acid treatment leads to greater rates of RNA synthesis, producing longer chains in unit time, suggests that one manifestation of auxin activity is in activation of RNA polymerase I (ribosomal RNA polymerase).
...
PMID:Greater Length of Ribonucleic Acid Synthesized by Chromatin-bound Polymerase from Auxin-treated Soybean Hypocotyls. 1665 38

Two DNA-dependent RNA polymerases (ribonucleoside triphosphate:RNA nucleotidyl transferase, EC 2.7.7.6) have been isolated from pea (Pisum sativum) seedlings. The enzymes were solubilized by sonication in high salt buffer and were separated by chromatography on diethylaminoethyl cellulose using a linear salt gradient. Polymerase I eluted at 0.10 m (NH(4))(2)SO(4), accounted for about 10% of the recovered activity and was completely insensitive to alpha-amanitin. Polymerase II eluted at 0.14 m (NH(4))(2)SO(4), accounted for the remaining 90% of recovered activity and was strongly inhibited by alpha-amanitin. Both enzymes preferred denatured to native DNA as template, both showed an absolute requirement of divalent cation, and both were sensitive to the ionic strength of the assay medium. The developing pea seedling seems a promising system for studies of possible changes in relative activities and roles of multiple RNA polymerases during eukaryotic development.
...
PMID:Separation and partial characterization of two ribonucleic Acid polymerases from pea seedlings. 1665 87

The relative levels of multiple RNA polymerases were determined in soybean (Glycine max L. var. Wayne) hypocotyl during various stages of development. The meristematic region of the hypocotyl contains more total polymerase activity per gram fresh weight and a greater proportion of polymerase I relative to II than the differentiated regions. The fully elongated tissue comprising the lower half of the hypocotyl contains mainly RNA polymerase II. The hook region contains a polymerase activity peak which is completely sensitive to alpha-amanitin and partially sensitive to rifamycin SV. This peak is not detectable in other regions of the hypocotyl. Polymerase I is reproducibly separated into a major and a minor component, both being resistant to alpha-amanitin. The two components elute at salt concentrations of 0.2 m and 0.23 m KCl, respectively, while the alpha-amanitin-sensitive polymerase (II) elutes at 0.3 m KCl. The polymerase activity peak which is detectable only in the hook region elutes at approximately 0.5 m KCl. Polymerase levels were also determined in water-stressed tissue and in tissue which was harvested after three days of growth instead of the usual four days.
...
PMID:Developmental Changes in Multiple Forms of Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase in Soybean Hypocotyl. 1665 25

A new method called promoter trapping was developed to purify promoter-protein complex using the c-jun promoter (-200+81) as a model, which was shown to have significant promoter activity. Polymerase chain reaction (PCR), lambda exonuclease digestion combined with (AC)(5)-Sepharose DNA affinity chromatography were used to produce c-jun promoter with a (GT)(5) tail at each 3' end. The intact promoter and different length pieces with one or two (GT)(5) tails had almost the same capacity to bind with (AC)(5)-Sepharose. In solution, tailed c-jun promoter (60 nM) and competitor poly dI:dC (30 ng/microl) was incubated with crude HEK293 nuclear extract to form a large protein-promoter complex, and the complex was then trapped by (AC)(5)-Sepharose by centrifugation or on a column. Compared with a popular alternative method, called here the immobilized promoter method, the products of promoter trapping were purer. The preinitiation complex purified by promoter trapping had the expected components including RNA polymerase II, TATA-box binding protein (TBP), TFIIF subunit RAP74, and transcription factor SP1, and transcribed RNA in vitro. Thus, the promoter trapping approach provides a useful tool for the purification and investigation of transcription complexes.
...
PMID:Promoter trapping of c-jun promoter-binding transcription factors. 1693 21

DNA polymerase epsilon co-operates with polymerases alpha and delta in the replicative DNA synthesis of eukaryotic cells. We describe here a specific physical interaction between DNA polymerase epsilon and RNA polymerase II, evidenced by reciprocal immunoprecipitation experiments. The interacting RNA polymerase II was the hyperphosphorylated IIO form implicated in transcriptional elongation, as inferred from (a) its reduced electrophoretic mobility that was lost upon phosphatase treatment, (b) correlation of the interaction with phosphorylation of Ser5 of the C-terminal domain heptapeptide repeat, and (c) the ability of C-terminal domain kinase inhibitors to abolish it. Polymerase epsilon was also shown to UV crosslink specifically alpha-amanitin-sensitive transcripts, unlike DNA polymerase alpha that crosslinked only to RNA-primed nascent DNA. Immunofluorescence microscopy revealed partial colocalization of RNA polymerase IIO and DNA polymerase epsilon, and immunoelectron microscopy revealed RNA polymerase IIO and DNA polymerase epsilon in defined nuclear clusters at various cell cycle stages. The RNA polymerase IIO-DNA polymerase epsilon complex did not relocalize to specific sites of DNA damage after focal UV damage. Their interaction was also independent of active DNA synthesis or defined cell cycle stage.
...
PMID:DNA polymerase epsilon associates with the elongating form of RNA polymerase II and nascent transcripts. 1721 75


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---