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)

We characterize a component of the E. coli bacterial nucleoid H1a, which accumulates in stationary phase. This protein, identical with the major component of a plasmid-protein complex previously isolated in our laboratory, has a pI close to 7.5. Acrylamide gel electrophoresis and sedimentation in sucrose gradient have shown that the protein H1a induces significant compaction into DNA. This compaction is equivalent to that observed in nucleosome core although it introduces only a slight change in linking number. In addition, the structural change induced in the lactose L8UV5 promoter by H1a results in the decrease in the kinetic of formation of the open complex with RNA polymerase.
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PMID:H1a, an E. coli DNA-binding protein which accumulates in stationary phase, strongly compacts DNA in vitro. 637

The rates of formation of RNA polymerase-promoter open complexes at the galactose P2 and lactose UV5 promoters of E. coli were studied using polyacrylamide gels to separate the heparin-resistant complexes from unbound DNA. Both the apparent rate and extent of reaction at these promoters are inhibited at excess RNA polymerase. This inhibition, which can be relieved by the addition of non-promoter DNA, is interpreted to be the result of occlusion of the promoter site by nonspecifically bound polymerase. Additionally, biphasic kinetics are observed at both gal P2 and lac UV5, but not at the PR promoter of phage lambda. This behavior disappears when the concentration of RNA polymerase in the binding reaction is less than that of the promoter fragment. It is proposed that at excess enzyme nonspecifically bound polymerase molecules sliding along the DNA may "bump" closed complexes from the promoter site thereby reducing the rate of open complex formation. Kinetics mechanisms quantifying both the occlusion and bumping phenomena are presented.
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PMID:Kinetics of RNA polymerase-promoter complex formation: effects of nonspecific DNA-protein interactions. 646 7

High-resolution gel electrophoresis has been used to detect and quantitate promoter-specific oligonucleotides produced during initiation of transcription in vitro at the lactose operon (lac) UV5 promoter. The resolved products are RNA species of various lengths which correspond to the initial lac mRNA sequence. Quantitation shows that many oligonucleotides can be formed per preinitiation complex, including species as long as hexanucleotide. Synthesis occurs without dissociation of the enzyme, as evidenced by levels of synthesis in the presence of heparin, a selective inhibitor of free RNA polymerase. Thus, RNA polymerase cycles at this promoter in vitro producing oligonucleotides reiteratively. In general, the yield of oligonucleotides decreases when the total concentration of all four substrates is increased or when a missing nucleoside triphosphate substrate is added. Nevertheless, oligonucleotide synthesis persists under all conditions tested. Strikingly, the dinucleotide always represents 50% of the total of all oligonucleotides, even when conditions are manipulated to cause a 100-fold variation in this total. This shows that, after formation of the first phosphodiester bond at the lac UV5 promoter, dissociation of the dinucleotide is as likely as formation of the second phosphodiester bond. As discussed above, after release of a small RNA, RNA polymerase may then begin another RNA chain, which is again subject to premature release. These considerations lead to a model in which RNA polymerase cycles to produce oligonucleotides during initiation of transcription at the lac UV5 promoter in vitro. Production of a long RNA transcript is then essentially an escape from this cycling reaction. The drug rifampicin, which drastically inhibits escape to produce RNA, limits, but dose not prevent, the cycling reaction.
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PMID:Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter. 699 2

We have constructed a cloning vector based on plasmid mini-F for use in Escherichia coli. Plasmid pZC320 consists of the ori-2 replication unit of F that confers very low copy number (lcn), and includes the sop partition functions to insure stable plasmid maintenance in the absence of selection. A multiple cloning site (MCS) containing 16 unique restriction sites is located within the 5' end of the lacZ alpha gene. Expression of lacZ alpha is under the control of the wild-type lactose operator/promoter (lacOP) region and is efficiently repressed by the lacI repressor. Clones containing inserts can be detected using the blue/white screen for beta-galactosidase (beta Gal). A T7 promoter allows transcription of cloned inserts in the presence of T7 RNA polymerase. We have demonstrated the use of this lcn vector for cloning the regulated tetracycline-resistance genes from Tn10, which confer only low-level resistance when present at high copy number.
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PMID:A versatile low-copy-number cloning vector derived from plasmid F. 759 Mar 21

Derivatives of the lac promoter (tac, pac, rac) belong to the strongest bacterial promoters which are frequently used for the induced overexpression of foreign genes in Escherichia coli. However, their use in fermentation processes is strongly restricted because of the high cost of the inducer iso-propyl-beta-D-thiogalactopyranoside (IPTG). The aim of this work was to investigate the possibility of using lac-derived promoters in high cell density processes resulting in a high yield of the induced recombinant protein if glucose is the main carbon and energy source. Lactose is tested as inducer of the main antigenic coat protein (VP1) of the foot and mouth disease (FMD) virus in a T7-RNA polymerase expression system. It was shown that lactose is able to induce the expression of the recombinant gene to an amount of the VP1 protein corresponding to 20% of the total cell protein.
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PMID:Efficient use of lactose for the lac promoter-controlled overexpression of the main antigenic protein of the foot and mouth disease virus in Escherichia coli under fed-batch fermentation conditions. 801 64

The DNA binding affinities of several gene-regulatory proteins, restriction endonucleases and the Escherichia coli RNA polymerase have previously been found to be dependent on the nature of the dominant buffer anion. To discover whether the E. coli cAMP receptor protein (CAP) exhibits a similar dependency, we measured its affinity for its primary lactose promoter binding site (lac site 1) in buffers in which the principal anion was chloride, phosphate, sulfate, acetate, or glutamate. We found that the affinity of CAP for lac site 1 is affected only slightly by changes in the dominant buffer anion. The binding of cAMP is similarly insensitive to buffer anion type, indicating that specific protein-anion interactions, if they occur, must be similar for the free and cAMP-bound forms of the protein. The effect of anion substitution on the ability of acrylamide to quench the intrinsic fluorescence of tryptophanyl residues of CAP is also small, suggesting that changes in buffer anion composition have minimal effect on the conformation of tryptophan-proximal regions of CAP. This conclusion is extended by the finding that anion substitution has a relatively small effect on the urea-concentration dependence of CAP denaturation. Taken together, these results support the notion that neither CAP nor CAP.cAMP nor the CAP.cAMP complex with lac promoter DNA interact selectively with anions present in the surrounding buffer. A possible role for this anion-insensitivity in the in vivo function of CAP is suggested.
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PMID:Effects of anions on the binding of the cAMP receptor protein to the lactose promoter. 838 49

A tightly regulated gene-expression system was developed using SP6 RNA polymerase (RpoSP6). The RpoSp6-encoding gene (rpoSP6) was inserted into a mini-F plasmid (mini-F) and expression was controlled by the lactose promoter (P(lac)) and operator (O(lac)) on the plasmid. Therefore, a controlled expression system for the target genes can easily be constructed in various host strains by co-transformation of the system plasmid pFSP6 with other vector plasmids containing the genes linked to the SP6 promoters (P(SP6)). Using the lac gene linked to P(SP6) as a reporter, we evaluated the regulation of expression in this system in various host strains. Low-level expression of lac was detected in Escherichia coli harboring this expression system when RpoSP6 was uninduced, although very low activities of beta-galactosidase (beta-Gal) were observed which were independent of the presence of pFSP6. This basal level of beta-Gal activity was possibly derived, because the P(SP6) element has very weak activity for E. coli RNA polymerase (Rpo). These results showed that RpoSP6 seemed to be produced at very low levels in uninduced cells. Beta-GAl activity increased about 18-32-fold when the expression of rpoSP6 was induced, as compared with the beta-Gal activity when uninduced. The tight regulation of this system is superior to that of other known systems and it has a considerable advantage for gene expression in E. coli.
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PMID:A tightly regulated expression system in Escherichia coli with SP6 RNA polymerase. 862 62

The lactose (lac) operon promoter is positively regulated by the catabolite gene activator-cyclic AMP complex (CAP) that binds to the DNA located 61.5 bp upstream of the transcription start site. Between the CAP binding site and the core promoter sequence is a 13-bp sequence (from -38 to -50 [the -45 region]). The possible roles of the -45 region in determining the CAP-independent level of lac expression and in the CAP activation process were studied by isolating and characterizing random multisite mutations. Only a small percentage of mutants have dramatic effects on lac promoter activity. Among the mutations that did affect expression, a 26-fold range in lac promoter activity in vivo was observed in the CAP-independent activity. The highest level of CAP-independent lac expression (13-fold the level of the wild-type lac promoter) correlated with changes in the -40 to -45 sequence and required an intact RNA polymerase alpha subunit for in vitro expression, as expected for an upstream DNA recognition element. Mutant promoters varied in their ability to be stimulated by CAP in vivo, with levels ranging from 2-fold to the wild-type level of 22-fold. Only a change of twofold in responsiveness to CAP could be attributed to direct DNA sequence effects. The -40 to -45 sequence-dependent enhancement of promoter activity and CAP stimulation of promoter activity did not act additively. The mutant promoters also displayed other characteristics, such as the activation of nascent promoter-like activities overlapping lac P1 and, in one case, replicon-dependent changes in promoter activity.
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PMID:The -45 region of the Escherichia coli lac promoter: CAP-dependent and CAP-independent transcription. 899 Feb 94

A monoclonal antibody raised to a Teladorsagia circumcincta 31-33 kDa doublet antigen was used to immunoscreen a T. circumcincta cDNA expression library. Sheep antibodies eluted from the proteins expressed by two clones immunopositive with the monoclonal antibody specifically recognised the doublet antigen on Western blots of third stage larval extract, confirming that these clones coded for the antigen. Database searches revealed high levels of similarity with beta-galactoside-binding lectin-like proteins (Ga1BPs or galectins) from Caenorhabditis elegans and Onchocerca volvulus. By analogy with these sequences, both T. circumcincta cDNA clones contain the full-length protein coding region. The native doublet proteins could be preferentially extracted from homogenates of third stage larvae with lactose and could be affinity purified on an asialofetuin column, confirming the identity of these bands as galectins. Reverse transcriptase-polymerase chain reaction amplification using a primer based on the C. elegans Spliced Leader SL1 sequence showed that the corresponding T. circumcincta mRNAs are also trans-spliced at their 5' ends. While there are considerable nucleotide differences between the two clones, the majority are located in the non-coding regions. Within the coding region there are 87 nucleotide differences but only three of these result in amino acid substitutions.
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PMID:cDNA cloning of galectins from third stage larvae of the parasitic nematode Teladorsagia circumcincta. 920 Jan 21

The use of lactose as inducer of foreign gene expression under control of the lac UV5 promoter was investigated in recombinant Escherichia coli. Chicken muscle troponin C (TnC) gene was transcripted by T7 RNA polymerase and expressed in bioreactor cultivations after a feed-forward controlled fed-batch growth phase. Cell concentrations of 22 g l-1 dry cell weight (DCW)--before induction started--were used to achieve a TnC content of 19.5% of total cell protein through an induction strategy that combined the addition of a specific lactose amount of 4.7 g g-1 DCW divided into three pulses and the addition of yeast extract (1 g l-1) together with the second and the third lactose pulses. The results presented suggest that the residual lactose concentration plays an important role on the production of the heterologous protein.
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PMID:Recombinant gene expression in Escherichia coli cultivation using lactose as inducer. 957 1


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