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)

To study the process of hormone action, we have developed an in vitro system utilizing minced oviduct from estrogen-treated chicks incubated in tissue culture medium. Progesterone added to the medium induced synthesis of a specific protein, avidin, that continued for up to 96 hr. During this period there was no increase in total oviduct protein, ovalbumin, or lysozyme, which suggests the specificity of the progesterone effect. The induction process was dependent on new protein synthesis, since cycloheximide inhibited the induction completely. Actinomycin D in doses that prevented nuclear RNA synthesis, but not general protein synthesis, inhibited avidin production 70-90%. Avidin synthesis was not affected by 5-fluorouracil. The rate of DNA synthesis examined by thymidine-(3)H pulse labeling was not stimulated during avidin induction. Hydroxyurea (an inhibitor of DNA synthesis) and colchicine (a mitotic inhibitor) did not prevent induction. Studies utilizing uridine-(3)H pulses showed an effect on rapdly labeled nuclear RNA coincident with induction. Nuclear RNA polymerase activity increased before avidin induction. Since avidin was the only new protein synthesized in response to progesterone, the early stimulation of nuclear RNA synthesis and RNA polymerase activity would suggest a mechanism of action for this steroid at the transcription level of protein synthesis.
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PMID:Studies on the mechanism of action of progesterone in regulation of the synthesis of specific protein. 563 49

Bacillus subtilis sporulating cells at stage III were fractionated into mother cell and forespore fractions by means of a lysozyme-detergent method. Three forms of DNA-dependent RNA polymerase enzymes, termed M sigma, F sigma, and F delta, in addition to core enzyme (alpha 2, beta', and beta) have been purified from the cell fractions. Enzymes M sigma and F sigma are present in the mother cell and forespore, respectively, and contain sigma factor of 55,000 daltons in addition to the core subunits. On the other hand, enzyme F delta is present specifically in the forespore and contains delta 1 factor of 28,000 daltons instead of the sigma factor. The amount of RNA polymerase in the forespore is about twice that in the mother cell. The enzymes M sigma and F sigma also differed in their elution profiled from DEAE-cellulose columns and in their heat stabilities indicating that the two sigma-containing holoenzyme forms may be different in their structural properties. The enzyme F delta transcribed B. subtilis DNA about 1.6 times more actively than enzyme F sigma, and the enzymes M sigma and F sigma transcribed the DNA about 2.2 times more actively than did core enzyme.
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PMID:Purification and properties of RNA polymerases from mother cells and forespores of sporulating cells of Bacillus subtilis. 679 62

We have determined the DNA sequence of a 770 bp Pst 1 fragment containing 450 nucleotides of the 5' flanking region of the chicken lysozyme gene. S1-nuclease mapping was performed to localize the 5' end of nuclear RNA containing lysozyme-specific sequences and of the mRNA. We present evidence that the 5' noncoding region of the chicken lysozyme mRNA is heterogeneous in length. The 5' termini of the different nRNAs map 29, 31 and 53 nucleotides upstream from their common initiation codon. The 5' ends of lysozyme-specific nuclear RNAs map at positions similar to that of the mRNA. AT-rich regions and sequences similar to the E. coli RNA polymerase recognition sequence are found around 30 and 70 nucleotides upstream from each of these 5' termini. The AT-rich regions differ, however, from the canonical Goldberg-Hogness box in that they do not contain the extremely conserved TATA sequence motif. Sequence comparison at the 5' end of the lysozyme, conalbumin and ovalbumin genes reveals only one region of partial homology, 140 nucleotides upstream from the mRNA start sites.
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PMID:Multiple mRNAs are generated from the chicken lysozyme gene. 728 17

Mutants of bacteriophage T7 RNA polymerase defective in functions other than transcription were sought by random chemical mutagenesis of the cloned gene and selection for inability to support the growth of a T7 mutant whose growth is dependent on T7 RNA polymerase supplied by the host cell. About half of the mutant clones appeared unable to make full-length T7 RNA polymerase, many of them producing a truncated protein. Among 116 mutants expressing full-length protein, two-thirds were severely impaired in transcription, but a surprisingly high one-third were able to direct significant transcription in vivo. Both types of mutation were distributed across much of the gene, as determined by a rapid genetic mapping procedure that allows the lethal mutation in each clone to be localized. One mutation (isolated twice) allowed normal gene expression but prevented the formation of mature ends of T7 DNA from concatemers, which normally happens during packaging into phage particles. Thirty-seven of the mutations appeared to increase the sensitivity of the polymerase to inhibition by T7 lysozyme; all were suppressed by mutations in the lysozyme gene, including one suppressor constructed to retain full amidase activity but to be unable to bind T7 RNA polymerase. The two lysozyme-hypersensitive polymerase mutants analyzed in detail showed premature cessation of transcription during infection. Early proteins and those late proteins specified by genes as far right in T7 DNA as genes 8-9 appeared to be produced normally, but expression of genes farther to the right was strongly depressed. DNA replication was depressed about 50% in one of these mutants and 90% in the other, even though the T7 replication proteins were made in normal amounts at the normal time.
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PMID:Isolation of transcriptionally active mutants of T7 RNA polymerase that do not support phage growth. 760 67

The lysozyme of bacteriophage T7 is a bifunctional protein that cuts amide bonds in the bacterial cell wall and binds to and inhibits transcription by T7 RNA polymerase. The structure of a mutant T7 lysozyme has been determined by x-ray crystallography and refined at 2.2-A resolution. The protein folds into an alpha/beta-sheet structure that has a prominent cleft. A zinc atom is located in the cleft, bound directly to three amino acids and, through a water molecule, to a fourth. Zinc is required for amidase activity but not for inhibition of T7 RNA polymerase. Alignment of the zinc ligands of T7 lysozyme with those of carboxypeptidase A and thermolysin suggests structural similarity among the catalytic sites for the amidase and these zinc proteases. Mutational analysis identified presumed catalytic residues for amidase activity within the cleft and a surface that appears to be the site of binding to T7 RNA polymerase. Binding of T7 RNA polymerase inhibits amidase activity.
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PMID:The structure of bacteriophage T7 lysozyme, a zinc amidase and an inhibitor of T7 RNA polymerase. 817 Oct 31

The use of microorganisms in the open environment would be of less concern if they were endowed with programmed self-destruction mechanisms. Here, we propose a new genetic design to increase the effectiveness of cell suicide systems. It ensures very tight control of the derepression of cell death by the combination of the bacteriophage T7 RNA polymerase-lysozyme system and an inducible synthesis of antisense RNA and the Escherichia coli LacI repressor. Functionality of this regulatory concept was tested by applying it to containment of Gram-negative bacteria, based on the conditional expression of the lethal Streptomyces avidinii streptavidin gene. Toxicity of streptavidin is derived from its exceptionally high binding affinity for an essential prosthetic group, D-biotin. The entire construct was designed to allow the soil bacterium Pseudomonas putida to survive only in the presence of aromatic hydrocarbons and their derivatives which it can degrade. Under favorable growth conditions, clones escaping killing appeared at frequencies of only 10(-7)-10(-8) per cell per generation. The general requirement for biotin through the living world should make streptavidin-based conditional lethal designs applicable to a broad range of containment strategies.
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PMID:A new approach for containment of microorganisms: dual control of streptavidin expression by antisense RNA and the T7 transcription system. 903 5

We have discovered that T7 RNA polymerase, purified to apparent homogeneity from overexpressing Escherichia coli cells, possesses a DNase and an RNase activity. Mutations in the active center of T7 RNA polymerase abolished or greatly decreased the nuclease activity. This nuclease activity is specific for single-stranded DNA and RNA oligonucleotides and does not manifest on double-stranded DNAs. Under the conditions of promoter-driven transcription on double-stranded DNA, no nuclease activity was observed. The nuclease attacks DNA oligonucleotides in mono- or dinucleotide steps. The nuclease is a 3' to 5' exonuclease leaving a 3'-OH end, and it degrades DNA oligonucleotides to a minimum size of 3 to 5 nucleotides. It is completely dependent on Mg2+. The T7 RNA polymerase-nuclease is inhibited by T7 lysozyme and heparin, although not completely. In the presence of rNTPs, the nuclease activity is suppressed but an unusual 3'-end-initiated polymerase activity is unmasked. RNA from isolated pre-elongation and elongation complexes arrested by a psoralen roadblock or naturally paused at the 3'-end of an oligonucleotide template exhibited evidence of nuclease activity. The nuclease activity of T7 RNA polymerase is unrelated to pyrophosphorolysis. We propose that the nuclease of T7 RNA polymerase acts only in arrested or paused elongation complexes, and that in combination with the unusual 3'-end polymerizing activity, causes heterogeneity in elongation complexes. Additionally, during normal transcription elongation, the kinetic balance between nuclease and polymerase is shifted in favor of polymerase.
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PMID:Nuclease activity of T7 RNA polymerase and the heterogeneity of transcription elongation complexes. 907 96

To investigate structure-function relationships in plant chitinases, we have developed a heterologous expression system for the 26 kDa endochitinase from Hordeum vulgare L. (barley). Escherichia coli cells harbouring the gene in a T7 RNA polymerase-based expression vector synthesized completely insoluble recombinant protein under standard induction conditions at 37 degrees C. However, a concentration of soluble recombinant protein of approx. 15 mg/l was achieved by inducing bacteria at low temperature (15 degrees C). Recombinant endochitinase was purified to homogeneity and shown to be structurally and functionally identical to the seed protein. An average of three disulphide bonds are present in the recombinant enzyme, consistent with the number found in the natural form. The seed and recombinant proteins showed the same specific activity towards a high-molecular-mass substrate and exhibited similar anti-fungal activity towards Tricoderma reesei. Site-directed mutagenesis was used to replace residues that are likely to be involved in the catalytic event, based on structural similarities with lysozyme and on sequence alignments with related chitinases. The Glu67-->Gln mutation resulted in a protein with undetectable activity, while the Glu89-->Gln mutation yielded an enzyme with 0. 25% of wild-type specific activity. This suggests that two acidic residues are essential for catalytic activity, similar to the situation with many other glycosyl hydrolases. Examination of conserved residues stretching into the proposed substrate binding cleft suggests that Asn124 also plays an important functional role.
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PMID:Heterologous expression and characterization of wild-type and mutant forms of a 26 kDa endochitinase from barley (Hordeum vulgare L.). 914 54

Members of the Bunyaviridae family mature by a budding process in the Golgi complex. The site of maturation is thought to be largely determined by the accumulation of the two spike glycoproteins, G1 and G2, in this organelle. Here we show that the signal for localizing the Uukuniemi virus (a phlebovirus) spike protein complex to the Golgi complex resides in the cytoplasmic tail of G1. We constructed chimeric proteins in which the ectodomain, transmembrane domain (TMD), and cytoplasmic tail (CT) of Uukuniemi virus G1 were exchanged with the corresponding domains of either vesicular stomatitis virus G protein (VSV G), chicken lysozyme, or CD4, all proteins readily transported to the plasma membrane. The chimeras were expressed in HeLa or BHK-21 cells by using either the T7 RNA polymerase-driven vaccinia virus system or the Semliki Forest virus system. The fate of the chimeric proteins was monitored by indirect immunofluorescence, and their localizations were compared by double labeling with markers specific for the Golgi complex. The results showed that the ectodomain and TMD (including the 10 flanking residues on either side of the membrane) of G1 played no apparent role in targeting chimeric proteins to the Golgi complex. Instead, all chimeras containing the CT of G1 were efficiently targeted to the Golgi complex and colocalized with mannosidase II, a Golgi-specific enzyme. Conversely, replacing the CT of G1 with that from VSV G resulted in the efficient transport of the chimeric protein to the cell surface. Progressive deletions of the G1 tail suggested that the Golgi retention signal maps to a region encompassing approximately residues 10 to 50, counting from the proposed border between the TMD and the tail. Both G1 and G2 were found to be acylated, as shown by incorporation of [3H]palmitate into the viral proteins. By mutational analyses of CD4-G1 chimeras, the sites for palmitylation were mapped to two closely spaced cysteine residues in the G1 tail. Changing either or both of these cysteines to alanine had no effect on the targeting of the chimeric protein to the Golgi complex.
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PMID:A retention signal necessary and sufficient for Golgi localization maps to the cytoplasmic tail of a Bunyaviridae (Uukuniemi virus) membrane glycoprotein. 915 65

We had earlier overproduced the transcription activator protein C of bacteriophage Mu in a phage-T7 expression system. Although we achieved a high level of overproduction, the expression was not consistent. This could be due to the leaky expression of T7 RNA polymerase in the uninduced state. Introduction of pLysS, a plasmid encoding T7 lysozyme, a natural inhibitor of T7 RNA polymerase, resulted in consistent, but extremely low production of the C protein. To overcome this problem, we have devised an artificial regulatory circuit to obtain stabilised, consistent overproduction of C protein. The C-binding site was cloned downstream from the transcription start point of T7 lys. Upon induction, the C protein produced binds to its site with a very high affinity, possibly acting as a transcriptional roadblock for lys. This would overcome the inhibitory effect of T7 lysozyme on T7 RNA polymerase.
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PMID:An artificial regulatory circuit for stable expression of DNA-binding proteins in a T7 expression system. 918 43


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