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 coding sequence for bacteriophage T7 RNA polymerase has been cloned and expressed under control of a cognate T7 promoter, a configuration referred to as an autogene. Cloning a T7 autogene in a derivative of plasmid pBR322 in Escherichia coli was achieved by a combination of blocking initiation at the T7 promoter with bound lac repressor and inhibiting the polymerase itself by T7 lysozyme. Neither type of inhibition by itself was sufficient to control the autogene. Upon unblocking the T7 promoter with added inducer. T7 RNA polymerase produced its own mRNA, leading to autocatalytic production of polymerase protein. T7 autogenes may be useful for developing high-level gene expression systems in a variety of cell types, with little if any need for the host cell RNA polymerase.
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PMID:Creation of a T7 autogene. Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter. 202 61

Plasmid pBR322 and its derivative containing strong promoter phi 10 of bacteriophage T7 RNA polymerase were used as vectors. A fragment of bacteriophage T7 DNA which was digested with two restriction endonucleases (AvaII and HaeIII) was cloned in the BamHI site of plasmid pBR322 and its derivative pAR951, respectively. The inserted DNA is a segment of 632 base pairs containing the complete coding sequence of both T7 gene 3.5 and weak promoter phi 3.8 for bacteriophage T7 RNA polymerase. The function of T7 gene 3.5 is known to code for bacteriophage T7 lysozyme. Transformants that carry the recombinant plasmid were tested for intracellular lysozyme by adding CHCl3. Both cloned strains produce active T7 lysozyme. The gene product, T7 3.5 protein, was analyzed by 10 -20% gradient polyacrylamide- SDS electrophoresis. The result showed that the expression of inserted T7 gene 3.5 in pBR322 derivative is stronger than that in pBR322.
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PMID:Cloning of the bacteriophage T7 lysozyme gene. 210 4

Matrix attachment regions (MARs) are DNA elements that dissect the genome into topologically separated domains by binding to a chromosomal skeleton. This study explored the putative influence of the MAR located 5' of the chicken lysozyme gene on expression of heterologous genes in heterologous cell systems. Expression of a construct with the chloramphenicol acetyltransferase (CAT) indicator gene controlled by the herpes simplex virus thymidine kinase promoter (TC) and a construct in which the same transcriptional unit is flanked by chicken lysozyme 5' MARs (MTCM) was assayed after stable transfection into rat fibroblasts. Median CAT activity per copy number in MTCM transfectants was elevated approximately 10-fold relative to that in TC transfectants. Total variation in normalized CAT activity decreased from more than 100-fold among TC transfectants to nearly 6-fold among MTCM transfectants. The steady-state level of transcripts and the relative rate of transcription were increased in MTCM transfectants, as shown by S1 nuclease and run-on transcription assays, respectively. The chicken lysozyme 5' MAR thus can confer elevated, less position-dependent expression on a heterologous promoter in cells of a different species by increasing the density of transcribing RNA polymerase molecules. MAR-mediated transcriptional enhancement suggests that MARs are important for gene expression and not just for DNA packaging.
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PMID:The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes. 232 53

RNA/RNA duplex formation involving the 5'untranslated region of a mRNA can efficiently block translation. Here we investigated the effect on translation of an RNA/RNA duplex between part of the coding region and sequences of the 3'untranslated region of lysozyme mRNA. A cDNA was constructed which contained 2 identical sequences of 150 nucleotides, one of which was an inverted repeat of the other. Cell-free transcription of this cDNA with T7 RNA polymerase resulted in a mRNA with an extended RNA/RNA duplex within the coding region. The presence of the double stranded structure was confirmed by the accessibility of complementary oligonucleotides to this region. mRNA was cleaved by RNaseH, endogenous to the wheat germ lysate, when hybridization of a complementary oligonucleotide occurred outside but not within the predicted double stranded structure. When this mRNA was translated in a cell-free wheat germ translation system, the translation product was found to be of the size of full-length prelysozyme and not arrested. We conclude that the extend of a secondary structure within the coding region of a mRNA does not restrict the ability of the ribosome to translate this mRNA efficiently. Our data are consistent with the presence of an activity unwinding RNA/RNA duplexes, which is associated with the translating ribosome.
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PMID:An extended RNA/RNA duplex structure within the coding region of mRNA does not block translational elongation. 245 39

1. Transcription from the chicken lysozyme gene domain and the density of RNA polymerase molecules was studied by incubating isolated hen oviduct nuclei in an in vitro transcription system with [alpha-32P]UTP and hybridizing the purified [32P]RNA with immobilized restriction fragments. 2. The labeled RNA hybridized most prominently to coding and flanking restriction fragments that contain repetitive sequences. 3. Relatively weak hybridization signals with a coding, single-copy, genomic fragment and cDNA fragments were detected using 5000 microCi [alpha-32P]UTP per ml of incubation mixture. 4. Flanking, non-repetitive fragments did not hybridize to the [32P]RNA. 5. The results show that transcription from repetitive sequences dominates over lysozyme gene specific transcription.
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PMID:Lysozyme gene specific transcription in isolated hen oviduct nuclei. 245 65

The selectivity of T7 RNA polymerase for its own promoters is used to direct all transcription and replication to bacteriophage T7 DNA during infection. We now find that T7 lysozyme, which is known to cut a bond in the peptidoglycan layer of the cell wall, forms a specific complex with T7 RNA polymerase and inhibits transcription. Mutations that weaken this interaction have been found in the coding sequence for T7 RNA polymerase; an affinity column containing wildtype polymerase selectively binds T7 lysozyme, but a similar column containing mutant polymerase does not. The lysozyme-polymerase interaction ensures a controlled burst of late transcription during infection, and could possibly have some direct role in replication and/or control of lysis.
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PMID:T7 lysozyme inhibits transcription by T7 RNA polymerase. 356 26

Synthetic RNAs coding for chicken lysozyme, calf preprochymosin and Xenopus globin were transcribed in vitro using Sp6 RNA polymerase. The effects of capping and adding a poly(A) tail on the stability, movement and translation of these RNAs in Xenopus oocytes was examined. Capping and polyadenylation increased stability of the transcripts, with at least 40% remaining intact 48 h after injection into oocytes. Capped poly(A)- transcripts moved more rapidly in oocytes than either capped poly(A)+ transcripts or naturally occurring mRNAs. The translational efficiency of most of the synthetic RNAs in oocytes increased with both capping and polyadenylation. The exception was one Xenopus globin transcript which had an unusual 3' end of 20As and 30Cs, where further polyadenylation decreased translational efficiency. Polyadenylation was essential for detectable expression of the synthetic RNAs in cultured cells, but decreased translation of the synthetic RNAs in vitro.
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PMID:The effect of capping and polyadenylation on the stability, movement and translation of synthetic messenger RNAs in Xenopus oocytes. 393 72

During nonpermissive infection by a T7 amber mutant in gene 1 (phage RNA polymerase-deficient), synthesis of the products of the phage genes 3 (endonuclease), 3, 5 (lysozyme), 5 (DNA polymerase), and 17 (serum blocking power) was shown to occur at about half the rate as during wild-type infection. This relatively high rate of expression of "late" genes (transcribed normally by the phage RNA polymerase) seems to be a general feature of all T7 mutants in gene 1 from our collection. In contrast, T3 gene 1 mutants and a T7 gene 1 mutant from another collection showed late protein synthesis at very reduced rates. Synthesis of the gene 3 endonuclease by T7 gene 1 mutants was very sensitive to the addition of rifampin 2 min after infection, conditions under which there was very little inhibition during wild-type infection. This supports the notion that late gene expression during nonpermissive infection by gene 1 mutants is dependent on the transcription of the T7 genome by the host RNA polymerase. In contrast to T3 gene 1 mutants, the T7 gene 1 mutants of our collection directed the synthesis of phage DNA during nonpermissive infection. This DNA accumulated as a material sedimenting faster than mature T7 DNA.
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PMID:Synthesis of bacteriophage-coded gene products during infection of Escherichia coli with amber mutants of T3 and T7 defective in gene 1. 457 63

The inability of T7 to develop in cells of Escherichia coli containing F(+) or substituted F' episomes is a result of the failure to synthesize late proteins; no in vivo translation of mRNA species synthesized by the T7 RNA polymerase occurs. Further experiments have been performed to measure the amount of late mRNA in T7-infected F'(PIF(+)) cells. (We have designated the property of phage inhibition of F factors as PIF; the wild-type episome is therefore F'[PIF(+)].) T7 late proteins were synthesized in vitro by using a system programed with RNA extracted from T7-infected F(-) and F'(PIF(+)) cells. The T7 lysozyme, product of gene 3.5, and the gene 10 head protein were assayed. The following results were obtained: (i) mRNA capable of supporting in vitro synthesis of lysozyme and the gene 10 head protein is present in T7-infected F'(PIF(+)) cells; (ii) lysozyme mRNA extracted from T7-infected F'(PIF(+)) cells is present at 70 to 75% of the level found in T7-infected F(-) cells; (iii) gene 10 mRNA is present at 35 to 78% of the level found in T7-infected F(-) cells. No in vivo synthesis of either lysozyme or gene 10 protein can be detected in T7-infected F'(PIF(+)) cells although normal synthesis of these proteins occurs in F(-) cells. These findings confirm that the block in T7 development in F'(PIF(+)) cells results from the failure to translate late classes of T7 RNA.
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PMID:Evidence for the presence of nontranslated T7 late mRNA in infected F'(PIF+) episome-containing cells. 458 54

Synthesis of many T7 proteins is prevented in F' episome-containing cells. In order to quantitate the degree of inhibition, we measured the activity of several T7 proteins in extracts prepared from T7-infected F(-) and F' cells and cells containing F factors mutant in phage inhibition [F'(PIF(-)2A) and F'(PIF(-)2A,2B)]. In addition, we were able to assign specific T7 proteins to the three translational units previously defined by polyacrylamide gel analysis of T7 proteins made in F(-) and episome-containing cells. After T7 infection, the presence of the wild-type F' (PIF(+)) episome led to greater than 90% inhibition of T7 DNA polymerase (product of gene 5), T7 lysozyme (gene 3.5), and gene 10 capsid protein synthesis. Nearly normal amounts of T7 RNA polymerase (gene 1) were made in these cells. T7 infection of cells containing the mutant F' (PIF(-)2A) episome led to normal synthesis of T7 RNA polymerase and T7 DNA polymerase; T7 lysozyme was synthesized at 30% of the maximal level in these cells; T7 gene 10 capsid protein synthesis was inhibited by 90%, and T7 DNA synthesis was arrested in these cells. T7 infection of cells containing the mutant F' (PIF(-)2A,2B) episome led to synthesis of normal levels of the enzymes assayed.
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PMID:T7 protein synthesis in F' episome-containing cells: assignment of specific proteins to three translational groups. 458 55


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