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 anti-sigma 70 factor of bacteriophage T4 is a 10-kDa (10K) protein which inhibits the sigma 70-directed initiation of transcription by Escherichia coli RNA polymerase holoenzyme. We have partially purified the anti-sigma 70 factor and obtained the sequence of a C-terminal peptide of this protein. Using reverse genetics, we have identified, at the end of the lysis gene t and downstream of an as yet unassigned phage T4 early promoter, an open reading frame encoding a 90-amino-acid protein with a predicted molecular weight of 10,590. This protein has been overproduced in a phage T7 expression system and partially purified. It shows a strong inhibitory activity towards sigma 70-directed transcription (by RNA polymerase holoenzyme), whereas it has no significant effect on sigma 70-independent transcription (by RNA polymerase core enzyme). At high ionic strength, this inhibition is fully antagonized by the neutral detergent Triton X-100. Our results corroborate the initial observations on the properties of the phage T4 10K anti-sigma 70 factor, and we therefore propose that the gene which we call asiA, identified in the present study, corresponds to the gene encoding this T4 transcriptional inhibitor.
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PMID:The asiA gene of bacteriophage T4 codes for the anti-sigma 70 protein. 841 14

Cells of two human follicular thyroid carcinoma cell lines (FTC133, FTC236) were stably transfected with a cDNA encoding the PDE4A cAMP-specific phosphodiesterase (PDE) splice variant RD1 (RNPDE4A1A) so as to generate the cloned cell lines, FTC133A and FTC236A. This allowed the expression of a novel rolipram-inhibited cAMP-specific PDE activity in these cells. Unlike the parent cell lines in which Ca2+/calmodulin caused a profound activation (approx. 3-4-fold) of homogenate PDE activity, no such stimulation was evident in the RD1-expressing cell lines, indicating loss of PDE1 activity. Reverse transcriptase-PCR analysis indicated that this was due to the down-regulation of the PDE1C isoform. The novel PDE4 activity in transfected cells was located exclusively in the membrane fraction, as was immunoreactive RD1. Low concentrations of the detergent Triton X-100, but not high NaCl concentrations, allowed RD1 to be solubilized. Laser scanning confocal immunofluorescence analyses identified RD1 immunoreactivity in a discrete perinuclear region of these RD1-expressing transfected cell lines. A similar pattern of labelling was observed using the antiserum Tex1, which specifically identified the Golgi apparatus. Treatment of FTC133A cells with the Golgi-perturbing agents monensin and brefeldin A led to a similar redistribution of immunoreactive species detected using both the Tex1 and anti-RD1 antisera. It is suggested that the PDE4A splice variant RD1 contains a membrane-association signal which allows the targeted expression of RD1 within the Golgi complex of these human follicular thyroid carcinoma cell lines.
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PMID:Intracellular localization of the PDE4A cAMP-specific phosphodiesterase splice variant RD1 (RNPDE4A1A) in stably transfected human thyroid carcinoma FTC cell lines. 900 17

In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type I signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pI of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum.
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PMID:Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2). 902 6

Pro-sigmaK is the inactive precursor of sigmaK, a mother cell-specific sigma factor responsible for the transcription of late sporulation genes of Bacillus subtilis. Upon subcellular fractionation, the majority of the pro-sigmaK was present in the membrane fraction. The rest of the pro-sigmaK was in a large complex that did not contain RNA polymerase core subunits. In contrast, the majority of the sigmaK was associated with core RNA polymerase. Virtually identical fractionation properties were observed when pro-sigmaE was analyzed. Pro-sigmaK was completely solubilized from the membrane fraction and the large complex by Triton X-100 and was partially solubilized from the membrane fraction by NaCl and KSCN. The membrane association of pro-sigmaK did not require spoIVF gene products, which appear to be located in the mother cell membrane that surrounds the forespore, and govern pro-sigmaK processing in the mother cell. Furthermore, pro-sigmaK associated with the membrane when overproduced in vegetative cells. Overproduction of pro-sigmaK in sporulating cells resulted in more pro-sigmaK in the membrane fraction. In agreement with the results of cell fractionation experiments, immunofluorescence microscopy showed that pro-sigmaK was localized to the mother cell membranes that surround the mother cell and the forespore in sporulating wild-type cells and mutant cells that do not process pro-sigmaK. Treatment of extracts with 0.6 M KCl appeared to free most of the pro-sigmaK and sigmaK from other cell constituents. After salt removal, sigmaK, but not pro-sigmaK, reassociated with exogenous core RNA polymerase to form holoenzyme. These results suggest that the prosequence inhibits RNA polymerase core binding and targets pro-sigmaK to the membrane, where it may interact with the processing machinery.
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PMID:The prosequence of pro-sigmaK promotes membrane association and inhibits RNA polymerase core binding. 957 96

Among the functions of the replicase of equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) are important viral enzyme activities such as proteases and the putative RNA polymerase and RNA helicase functions. The replicase is expressed in the form of two polyproteins (open reading frame 1a [ORF1a] and ORF1ab), which are processed into 12 nonstructural proteins by three viral proteases. In immunofluorescence assays, the majority of these cleavage products localized to the perinuclear region of the cell. A dense granular and vesicular staining was observed, which strongly suggested membrane association. By using confocal microscopy and double-label immunofluorescence, the distribution of the EAV replicase was shown to overlap with that of PDI, a resident protein of the endoplasmic reticulum and intermediate compartment. An in situ labeling of nascent viral RNA with bromo-UTP demonstrated that the membrane-bound complex in which the replicase subunits accumulate is indeed the site of viral RNA synthesis. A number of ORF1a-encoded hydrophobic domains were postulated to be involved in the membrane association of the arterivirus replication complex. By using various biochemical methods (Triton X-114 extraction, membrane purification, and sodium carbonate treatment), replicase subunits containing these domains were shown to behave as integral membrane proteins and to be membrane associated in infected cells. Thus, contribution to the formation of a membrane-bound scaffold for the viral replication-transcription complex appears to be an important novel function for the arterivirus ORF1a replicase polyprotein.
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PMID:ORF1a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex. 965 16

The XPA protein is essential for both of the known modes of nucleotide excision repair (NER) in human cells: transcription-coupled repair (TCR) and global genome repair (GGR). In TCR, this protein is thought to be recruited to lesion sites in DNA at which RNA polymerase II is blocked and in GGR, by direct recognition of damages by repair protein complex containing XPC/HR23B or DNA damage-binding protein. However, details of the recruitment of the XPA protein in vivo are unknown. It was shown earlier that a portion of another NER protein, PCNA, which is completely extractable from non-S-phase mammalian nuclei, becomes insoluble after ultraviolet (UV) light irradiation and cannot be extracted by methanol or buffer containing Triton X-100. In the present study, we have found that UV light irradiation of human or Chinese hamster cells leads to decrease of extractability of the XPA protein by Triton X-100. Maximal insolubilization of the XPA protein is observed 1-4 h after irradiation but it is not detectable by 22 h. This effect is dose-dependent for UV light from 2.5 to 15 J/m(2) and is unaffected by the pre-treatment of cells with sodium butyrate, an inhibitor of histone deacetylation. The UV light-induced insolubilization of the XPA protein was also observed in two lines of Cockayne syndrome complementation group A cells, indicating that the effect is not dependent upon TCR. The results are discussed in relation to possible mechanisms of NER.
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PMID:Reduced extractability of the XPA DNA repair protein in ultraviolet light-irradiated mammalian cells. 1060 36

Abdominal aortic aneurysm (AAA) is a common disease of human aorta with increased incidence. It is a complication to atherosclerosis and it is closely associated with alterations in extracellular macromolecules. In this study, the levels of mRNA for versican--the major extracellular arterial proteoglycan (PG)--present in AAA and normal aortas were evaluated by reverse-transcriptase polymerase chain reaction. The concentration of versican was also examined in corresponding tissue samples. Versican was almost completely extracted with 4 M guanidine hydrochloride in the presence of Triton X-100, isolated by chromatography on DEAE-Sephacel and characterized using treatment with specific chondro-/dermato-lyases and agarose gel electrophoresis. Versican localization in tissue as well as the variation and distribution of smooth muscle cells (SMCs) and macrophages were also investigated immunohistochemically. The mRNAs coding for versican isoforms V(0) and V(1) were identified in both tissues, whereas V(2) was absent. The expression of V(0) was decreased 40% in aneurysmal vessel wall, whereas that for V(1) remained constant. This change was simultaneous with a significant decrease in versican concentration by 89%. In normal aortas, most versican was seen in the intima, whereas in AAA, this layer is characterized by advanced atherosclerotic lesion, rich in lipids and macrophages but poor in versican. The decreased transcription and the still lower amount of versican in the AAA may correlate to (i) a decrease in density of SMCs, these cells being the major source of versican in aorta, and (ii) the presence of macrophages, which may induce versican degradation and modulate versican synthesis. It is proposed that the decreased synthesis and increased degradation of versican, particularly of isoform V(0), and the resulting low concentration in the intima are crucial factors contributing to the altered viscoelastic and compressive properties and thereby to the deformity and dilatation of aorta.
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PMID:Human abdominal aortic aneurysm is characterized by decreased versican concentration and specific downregulation of versican isoform V(0). 1116 69

A gene coding for an esterase (PsEst1, 1911bp in length) of the psychrotrophic bacterium Pseudomonas sp. B11-1 isolated from Alaskan soil was cloned and sequenced. The deduced amino acid sequence revealed a protein of 637 amino acid residues with a molecular mass of 69 kDa. Although the expression product, PsEst1, showed no appreciable sequence similarity (less than 15% identity) to any known proteins with the established biochemical functions, it is expected to be related to the alpha/beta hydrolase superfamily because it shared sequence motifs that have been identified with this superfamily. For example, a unique 'nucleophilic elbow' motif, -Gly(36)-Asp-Ser-Leu-Asn(40)-, was identified, and Ser(38) was predicted to constitute a catalytic triad with Asp(162) and His(303). PsEst1 was overexpressed using a T7 RNA polymerase transcription (pET21a) system in the Escherichia coli BL21(DE3) cells as an inclusion body. A soluble denatured form of the enzyme was purified to homogeneity in the presence of 8M urea, and the catalytically active form of the enzyme could be obtained by subsequent removal of urea by dialysis, where the addition of 0.1% Triton X-100 was essential for the efficient renaturation of the enzyme. To our knowledge, this was the first example of the successful renaturation of the recombinant cold-adapted enzyme. The enzyme efficiently hydrolyzed vinyl and aryl esters with the C4-C6 acyl chain. The activation energy of the enzymatic p-nitrophenyl butyrate hydrolysis (20.1 kcal/mol at 10 degrees C) was significantly lower than the value (79.9 kcal/mol) of the mesophilic lipase. It was observed that the K(m) values for p-nitrophenyl butyrate in the growth temperature range of strain B11-1 (5-15 degrees C) were lower than those at higher temperatures.
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PMID:Cloning, heterologous expression, renaturation, and characterization of a cold-adapted esterase with unique primary structure from a psychrotroph Pseudomonas sp. strain B11-1. 1288 Jul 65

An endogenous cowpea mosaic virus (CPMV) RNA-protein complex (CPMV replication complex) capable of elongating in vitro preexisting nascent chains to full-length viral RNAs has been solubilized from the membrane fraction of CPMV-infected cowpea leaves using Triton X-100 and purified by Sepharose 2B chromatography and glycerol gradient centrifugation in the presence of Triton X-100. Analysis of the polypeptide composition of the complex by NaDod-SO(4)/PAGE and silver staining revealed major polypeptides with molecular masses of 110, 68, and 57 kilodaltons (kDa), among which the 110-kDa polypeptide was consistently found to cosediment precisely with the RNA polymerase activity. Using antisera to specific viral proteins, we found the 110-kDa polypeptide to be the only known viral polypeptide associated with the RNA replication complex, the 68- and 57-kDa polypeptides being most probably host-specific. The host-encoded 130-kDa monomeric RNA-dependent RNA polymerase, which is known to be stimulated in CPMV-infected cowpea leaves, did not copurify with the virus-specific RNA polymerase complex. Our results dispute the hypothesis that plant viral RNA replication may be mediated by the RNA-dependent RNA polymerase of uninfected plants. We tentatively conclude that the 110-kDa polypeptide encoded by the bottom component RNA of CPMV constitutes the core of the CPMV RNA replication complex.
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PMID:Purification of cowpea mosaic virus RNA replication complex: Identification of a virus-encoded 110,000-dalton polypeptide responsible for RNA chain elongation. 1659 43

When pea etioplast preparations were treated with Triton X-100, the membranes disappeared, the pigments were solubilized, and the organelles appeared to disintegrate. Low speed centrifugation (2000g) of the preparations following treatment with Triton X-100 resulted in a pellet which contained considerable quantities of plastid material. This included RNA polymerase and DNA polymerase activity, much of the DNA, about 30% of the RNA, and 50% of the protein of the washed plastid. The amount of RNA polymerase and DNA polymerase activity associated with the low speed pellet was dependent on the pH during Triton treatment. Significant quantities of the RNA polymerase activity of chloroplasts from spinach, peas, and tobacco were also recovered in the pellet after Triton treatment.
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PMID:Some effects of triton x-100 on pea etioplasts. 1665 82


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