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)

Bacillus subtilis ferrochelatase (EC 4.99.1.1), the final enzyme in protoheme IX biosynthesis, was produced with an inducible T7 RNA polymerase expression system in Escherichia coli and purified from the soluble cell fraction. It was crystallized from polyethylene glycol solution using the microseeding technique. The crystals diffract to a minimum Bragg spacing of 2.1 A. The space group is P4(2) with unit cell dimensions a = b = 50.2 A, c = 120.1 A.
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PMID:Purification, crystallization, and preliminary X-ray analysis of Bacillus subtilis ferrochelatase. 874 60

We modified a cell-free coupled transcription/translation system from Escherichia coli with the T7 phage RNA polymerase, and achieved a productivity as high as 0.4 mg protein/ml reaction mixture. First, we found that the optimal concentrations of phosphoenolpyruvate and poly(ethylene glycol) are interdependent; higher concentrations of the former should be used at higher concentrations of the latter. Second, the use of a condensed 30000 x g cell extract, in place of the conventional one, significantly increased the initial rate of protein synthesis. This phenomenon was demonstrated to be due to a reason other than elimination of inhibitory molecule(s) from the extract. For this system with the condensed extract, the phosphoenolpyruvate and poly(ethylene glycol) concentrations were again co-optimized, resulting in production of chloramphenicol acetyltransferase at a productivity of 0.3 mg/ml. Finally, the productivity was further increased up to 0.4 mg/ml, by supplementation of the pool of amino acids. This improved cell-free protein synthesis system is superior in productivity to any other cell-free systems reported so far, including the continuous-flow cell-free system.
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PMID:A highly efficient cell-free protein synthesis system from Escherichia coli. 877 39

Nucleotide excision repair (NER) and RNA polymerase II transcription are cellular processes that require the transcription/NER factor TFIIH. We have developed a whole cell extract from the yeast Saccharomyces cerevisiae that simultaneously supports both NER and RNA polymerase II transcription of independent substrates. NER activity in the yeast whole cell extract was readily detected in the absence of further supplementation but was stimulated in the presence of overexpressed Rad2 protein. The repair of N-acetyl-2-aminofluorene (AAF)-damaged DNA was dependent on RAD genes required for NER and deficient repair in rad mutant extracts was complemented by mixing different mutant extracts or by purified Rad proteins. Both the NER and transcription activities were stimulated by 5% polyethylene glycol in the whole cell extracts. Transcription activity from the template pCYC1G- was not affected by the presence of uracil-containing or AAF-damaged pUC18 DNA, which was expected to result in base excision repair (BER) and NER, respectively. An in vitro condition was defined that supported simultaneous NER and transcription independently in different substrates in the yeast whole cell extracts.
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PMID:A yeast whole cell extract supports nucleotide excision repair and RNA polymerase II transcription in vitro. 881 36

Nucleoids from Escherichia coli were isolated in the presence of spermidine at low salt concentrations. The nucleoids denature at relatively low temperatures or salt concentrations, yielding broad slowly sedimenting zones and/or macroscopic aggregates upon sucrose gradient centrifugation. Denaturation is accompanied by a loss of a characteristically compact shape as visualized by light and electron microscopy. Addition of polyethylene glycol or dextran prevents these changes, extending the range of stability of the isolated nucleoids to temperatures and ionic conditions like those which commonly occur in vivo. The effects of the polymers are consistent with stabilization by macromolecular crowding. Enzymatic digestion of the nucleoid DNA primarily releases three small proteins (H-NS, FIS, and HU) and RNA polymerase, as well as residual lysozyme from the cell lysis procedure. If isolated nucleoids are extracted with elevated salt concentrations under crowded, stabilized conditions, two of the proteins (HU and lysozyme) are efficiently removed and the compact form of the nucleoids is retained. These extracted nucleoids maintain their compact form upon reisolation into the initial uncrowded low-salt medium, indicating that HU, the most common "histone-like" protein of E. coli, is not a necessary component for maintaining compaction in these preparations.
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PMID:Stabilization of compact spermidine nucleoids from Escherichia coli under crowded conditions: implications for in vivo nucleoid structure. 924 71

Combining a system for binding proteins to surfaces (Sigal, G. B., C. Bamdad, A. Barberis, J. Strominger, and G. M. Whitesides. 1996. Anal. Chem. 68:490-497) with a method for making ultraflat gold surfaces (Hegner, M., P. Wagner, and G. Semenza. 1993. Surface Sci. 291:39-46 1993) has enabled single, oriented, active Escherichia coli RNA polymerase (RNAP) molecules to be imaged under aqueous buffer using tapping-mode atomic force microscopy (AFM). Recombinant RNAP molecules containing histidine tags (hisRNAP) on the C-terminus were specifically immobilized on ultraflat gold via a mixed monolayer of two different omega-functionalized alkanethiols. One alkanethiol was terminated in an ethylene-glycol (EG) group, which resists protein adsorption, and the other was terminated in an N-nitrilotriacetic acid (NTA) group, which binds the histidine tag through two coordination sites with a nickel ion. AFM images showed that these two alkanethiols phase-segregate. Specific binding of the hisRNAP molecules was followed in situ by injecting proteins directly into the AFM fluid cell. The activity of the hisRNAP bound to the NTA groups was confirmed with a 42-base circular single-stranded DNA template (rolling circle), which the RNAP uses to produce huge RNA transcripts. These transcripts were imaged in air after the samples were rinsed and dried, since RNA also has low affinity for the EG-thiol and cannot be imaged under the buffers we used.
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PMID:Oriented, active Escherichia coli RNA polymerase: an atomic force microscope study. 991 34

The Rhodobacter capsulatus BchI protein is one of three subunits of Mg chelatase, the enzyme which catalyzes the first committed step of chlorophyll and bacteriochlorophyll biosynthesis. The BchI protein was produced with an inducible T7 RNA polymerase expression system in Escherichia coli. The protein was purified from the soluble cell-extract fraction and crystallized from polyethylene glycol solution. The crystals diffract to a minimum Bragg spacing of 2.1 A. The space group is P63 with unit-cell dimensions a = b = 90.6, c = 84.1 A.
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PMID:Crystallization and preliminary X-ray analysis of the Rhodobacter capsulatus magnesium chelatase BchI subunit. 1008 53

We give a detailed account on the enzymatic synthesis of RNA conjugates by T7 RNA polymerase using modified initiator nucleotides during transcription. Following two different routes, ternary conjugates of guanosine-5'-monophosphate, poly(ethylene glycol), and anthracene were synthesized via phosphoramidite intermediates and characterized by a variety of spectroscopic techniques. Up to a degree of polymerization nPEG of about 17, these conjugates were efficiently incorporated into RNA by T7 RNA polymerase at the 5'-termini, thereby giving access to RNA conjugates required for biochemical studies as well as for the exploration of the catalytic potential of ribonucleic acids. The resulting conjugates are intact and functional.
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PMID:Ternary conjugates of guanosine monophosphate as initiator nucleotides for the enzymatic synthesis of 5'-modified RNAs. 1034 66

The plastid encoded RNA polymerase subunit genes rpoA, B and C1 of tobacco were disrupted individually by PEG-mediated plastid transformation. The resulting off-white mutant phenotype is identical for inactivation of the different genes. The mutants pass through a normal ontogenetic cycle including flower formation and production of fertile seeds. Their plastids reveal a poorly developed internal membrane system consisting of large vesicles and, occasionally, flattened membranes, reminiscent of stacked thylakoids. The rpo- material is capable of synthesising pigments and lipids, similar in composition but at lower amounts than the wild-type. Western analysis demonstrates that plastids contain nuclear-coded stroma and thylakoid polypeptides including terminally processed lumenal components of the Sec but not of the DeltapH thylakoid translocation machineries. Components using the latter route accumulate as intermediates. In striking contrast, polypeptides involved in photosynthesis encoded by plastid genes could not be detected by Western analysis, although transcription of plastid genes, including the rrn operon, by the plastid RNA polymerase of nuclear origin is found as expected. Remarkably, ultrastructural, sedimentation and Northern analyses as well as pulse experiments suggest that rpo- plastids contain functional ribosomes. The detection of the plastid-encoded ribosomal protein Rpl2 is consistent with these results. The findings demonstrate that the consequences of rpo gene disruption, and implicitly the integration of the two plastid polymerase types into the entire cellular context, are considerably more complex than presently assumed.
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PMID:Targeted disruption of the plastid RNA polymerase genes rpoA, B and C1: molecular biology, biochemistry and ultrastructure. 1041 98

Osteoblasts express protease-activated receptor-1 (PAR-1), which is activated by thrombin or by synthetic peptides corresponding to the new "tethered ligand" N-terminus of PAR-1 created by receptor cleavage. Both thrombin and human PAR-1-activating peptide stimulate an elevation of [Ca2+]i in the human SaOS-2 osteoblast-like cell line, but the peptide stimulates receptor-mediated Ca+ entry, whereas thrombin does not. Stimulation of proliferation in rat primary osteoblast-like cells is greater in response to rat PAR-1-activating peptide than to thrombin. Because the PAR-1-activating peptides are now known to activate PAR-2, the current study was undertaken to investigate whether osteoblasts express this receptor and, if so, whether this could account for the observed discrepancies between responses of osteoblasts to thrombin and to PAR-1-activating peptides. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemical studies demonstrated expression of PAR-2 by primary cultures of rat calvarial osteoblast-like cells. In immunohistochemical studies of embryonic mouse bones, osteoblasts showed positive staining for the presence of PAR-2. Activators of PAR-2 include trypsin, mast cell tryptase, gingipain-R, and synthetic peptides corresponding to the PAR-2 tethered ligand sequence. Treatment of primary rat osteoblast-like cells with rat PAR-2-activating peptide (SLIGRL), or SaOS-2 cells with human PAR-2-activating peptide (SLIGKV), caused a dose-dependent increase in [Ca2+]i. Trypsin or gingipain-R also induced an increase in intracellular calcium concentration, and caused reciprocal cross desensitization. Activators of PAR-2 caused a sharp peak in [Ca2+]i followed by a sustained plateau; [Ca2+]i returned to baseline levels upon treatment with ethylene-glycol tetraacetic acid (EGTA). Treatment of rat osteoblast-like cells in vitro with SLIGRL did not affect thymidine incorporation or endogenous alkaline phosphatase activity. The results presented here demonstrate that osteoblasts express PAR-2, and that such expression is able to account for the observed discrepancies between thrombin and PAR-1-activating peptides in their ability to evoke calcium entry, but not proliferative responses.
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PMID:Expression of protease-activated receptor-2 by osteoblasts. 1061 51

The detailed syntheses of the sulfhydryl-modified guanosine monophosphates 5'-deoxy-5'-thioguanosine-5'-monophosphorothioate (GSMP), O-[omega-sulfhydryl-tetra(ethylene glycol)]-O-(5'-guanosine) monophosphate (5'-HS-PEG4-GMP), and O-[omega-sulfhydryl-di(ethylene glycol)]-O-(5'-guanosine) monophosphate (5'-HS-PEG2-GMP) are reported. Transcription reactions employing GSMP, 5'-HS-PEG4-GMP, or 5'-HS-PEG2-GMP as the initiator nucleotide for T7 RNA polymerase introduce a thiol group at the 5'-end of RNA. The efficiency of thiol incorporation at the 5'-terminus of modified RNA compounds was assayed with three different thiol-reactive biotinylated reagents followed by streptavidin gel-shift methods. The transcription efficiency with various ratios of GTP to 5'-HS-PEG2-GMP was explored by reaction with a sulfhydryl-reactive maleimide-conjugated protein. This is an efficient method to incorporate enzymatically a thiol group into the 5'-end of RNA.
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PMID:5'-sulfhydryl-modified RNA: initiator synthesis, in vitro transcription, and enzymatic incorporation. 1171 85


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