Biomedical subjects

C C Richardson

Publications and source records attributed to C C Richardson.

At least 91 records · Page 5Linked to original sources

PubMed1987

Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7.

Bacteriophage T7 gene 5 protein has been purified to apparent homogeneity from cells overexpressing its gene several hundred-fold. Gene 5 protein is a DNA polymerase with low processivity; it dissociates from the primer-template after catalyzing the incorporation of 1-50 nucleotides, depending on the salt concentration. Escherichia coli thioredoxin, a host protein that is tightly associated with the gene 5 protein in phage-infected cells, is not required for this activity. Thioredoxin acts as an accessory protein to bestow processivity on the polymerizing reaction; DNA synthesis catalyzed by the gene 5 protein-thioredoxin complex on a single-stranded DNA template can polymerize thousands of nucleotides without dissociation. Conditions that increase the stability of secondary structures in the template (i.e., low temperature or high ionic strength) decrease the processivity. E. coli single-stranded DNA-binding protein stimulates both the rate of elongation and the processivity of the gene 5 protein-thioredoxin complex.

DNA-Directed DNA Polymerase↗

PubMed1987

Escherichia coli thioredoxin stabilizes complexes of bacteriophage T7 DNA polymerase and primed templates.

The DNA polymerase activity induced after bacteriophage T7 infection of Escherichia coli is found in a complex of two proteins, the T7 gene 5 protein and a host protein, thioredoxin. Gene 5 protein is a DNA polymerase and a 3' to 5' exonuclease. Thioredoxin binds tightly to the gene 5 protein and increases the processivity of polymerization some 1000-fold. Gene 5 protein forms a short-lived complex with the primer-template, poly(dA).oligo(dT), in the absence of Mg2+ and nucleotides. Thioredoxin increases the half-life of the preformed primer-template-polymerase complex from less than a second to approximately 5 min. The dissociation is accelerated by excess single-stranded DNA in an apparent second order reaction, indicating direct transfer of polymerase between DNA fragments. Thioredoxin also reduces the equilibrium dissociation constant, Kd, of the gene 5 protein -poly(dA).oligo(dT) complex 20- to 80-fold. The salt dependence of Kd indicates that thioredoxin stabilizes the primer-template-polymerase complex mainly through additional charge-charge interactions, increasing the estimated number of interactions from 2 to 7. The affinity of gene 5 protein for single-stranded DNA is at least 1000-fold higher than for double-stranded DNA and is little affected by thioredoxin. Under conditions of steady state synthesis the effect of thioredoxin on the polymerization rate is determined by two competing factors, an increase in processivity and a decrease of the dissociation rate of polymerase and replicated template.

Bacterial Proteins↗

PubMed1987

Selective oxidation of the exonuclease domain of bacteriophage T7 DNA polymerase.

Bacteriophage T7 DNA polymerase, the product of gene 5 of the phage, has both polymerase and single-and double-stranded DNA 3'-to 5'-exonuclease activities. The exonuclease activities can be inactivated selectively by an oxidation reaction that requires molecular oxygen, a reducing agent, and iron at a concentration less than or equimolar to that of the gene 5 protein. Both exonuclease activities can be diminished by several thousandfold, with only a small decline in the polymerase activity. Escherichia coli thioredoxin, an accessory protein that binds tightly to the gene 5 protein and increases the processivity of the polymerization reaction, has no effect on the rate of oxidation. We propose that iron binds specifically to the exonuclease domain and, in the presence of molecular oxygen and a reducing agent, generates reactive oxygen species that selectively modify amino acid residues essential for the exonuclease activities.

Bacterial Proteins↗

PubMed1987

Gene 18 protein of bacteriophage T7. Overproduction, purification, and characterization.

Genetic and physical analyses indicate that gene 18 protein of bacteriophage T7 is essential for packaging of T7 DNA. T7 DNA is replicated via linear intermediates, culminating in the formation of concatemers many genomes in length which are then packaged into capsids. In infections with phage carrying amber mutations in gene 18, development is blocked at the concatemer stage. Biochemical studies on the role of gene 18 protein in concatemer processing and DNA packaging have been hampered by its low level of expression of gene 18 during T7 infections. We have cloned gene 18 on a plasmid downstream from the bacteriophage lambda PL promoter controlled by the temperature-sensitive lambda repressor encoded by c 1857. Thermal induction leads to the expression of the 10,000-Da gene 18 protein to the extent of approximately 10% of the total protein after 2 h. The overexpressed gene 18 protein is susceptible to proteolytic degradation, a condition that can be alleviated by expression in an Escherichia coli strain carrying the lon100 deletion which reduces production of protease La. Extracts of induced cells will complement an extract of T7-infected cells lacking gene 18 protein for packaging of exogenous T7 DNA. The assay has been used to monitor the purification of gene 18 protein to essential homogeneity. The identity of the purified protein has been confirmed by sequencing of the N terminus. Gel filtration analysis suggests that the native protein is an octomer. Treatment of gene 18 protein with 3 M guanidine hydrochloride denatures it to a monomer. Removal of the denaturing agent by dialysis regenerates the octomeric structure and the ability to complement packaging extracts.

Chromatography, Gel↗

PubMed1987

Processing of concatemers of bacteriophage T7 DNA in vitro.

The T7 chromosome is a double-stranded linear DNA molecule flanked by direct terminal repeats or so-called terminal redundancies. Late in infection bacteriophage T7 DNA accumulates in the form of concatemers, molecules that are comprised of T7 chromosomes joined in a head to tail arrangement through shared terminal redundancies. To elucidate the molecular mechanisms of concatemer processing, we have developed extracts that process concatemeric DNA. The in vitro system consists of an extract of phage T7-infected cells that provides all T7 gene products and minimal levels of endogenous concatemeric DNA. Processing is analyzed using a linear 32P-labeled substrate containing the concatemeric joint. T7 gene products required for in vitro processing can be divided into two groups; one group is essential for concatemer processing, and the other is required for the production of full length left-hand ends. The products of genes 8 (prohead protein), 9 (scaffolding protein), and 19 (DNA maturation) along with gene 18 protein are essential, indicating that capsids are required for processing. In extracts lacking one or more of the products of genes 2 (Escherichia coli RNA polymerase inhibitor), 5 (DNA polymerase), and 6 (exonuclease), full length right-hand ends are produced. However, the left-hand ends produced are truncated, lacking at least 160 base pairs, the length of the terminal redundancy. Gene 3 endonuclease, required for concatemer processing in vivo, is not required in this system. Both the full length left- and right-hand ends produced by the processing reaction are protected from DNase I digestion, suggesting that processing of the concatemeric joint substrate is accompanied by packaging.

DNA, Viral↗

PubMed1987

Purification and characterization of the gene 1.2 protein of bacteriophage T7.

Gene 1.2 of bacteriophage T7, located near the primary origin of DNA replication at position 15.37 on the T7 chromosome, encodes a 10,059-dalton protein that is essential for growth on Escherichia coli optA1 strains (Saito, H., and Richardson, C. C. (1981) J. Virol. 37, 343-351). In the absence of the T7 1.2 and E. coli optA gene products, the degradation of E. coli DNA proceeds normally, and T7 DNA synthesis is initiated at the primary origin. However, T7 DNA synthesis ceases prematurely and the newly synthesized DNA is degraded; no viable phage particles are released. The gene 1.2 protein has been purified to apparent homogeneity from cells in which the cloned 1.2 gene is overexpressed. Purification of the [35S] methionine-labeled protein was followed by monitoring the radioactivity of the protein and by gel electrophoresis. The purified protein has been identified as the product of gene 1.2 on the basis of molecular weight and partial amino acid sequence. We have found that extracts of E. coli optA1 cells infected with T7 gene 1.2 mutants are defective in packaging exogenous T7 DNA when such extracts are prepared late in infection. Purified gene 1.2 protein restores packaging activity to these defective extracts, thus providing a biological assay for gene 1.2 protein. No specific enzymatic activity has been found associated with the purified gene 1.2 protein.

Amino Acid Sequence↗

PubMed1987

Gene 1.2 protein of bacteriophage T7. Effect on deoxyribonucleotide pools.

The gene 1.2 protein of bacteriophage T7, a protein required for phage T7 growth on Escherichia coli optA1 strains, has been purified to apparent homogeneity and shown to restore DNA packaging activity of extracts prepared from E. coli optA1 cells infected with T7 gene 1.2 mutants (Myers, J. A., Beauchamp, B. B., White, J. H., and Richardson, C. C. (1987) J. Biol. Chem. 262, 5280-5287). After infection of E. coli optA1 by T7 gene 1.2 mutant phage, under conditions where phage DNA synthesis is blocked, the intracellular pools of dATP, dTTP, and dCTP increase 10-40-fold, similar to the increase observed in an infection with wild-type T7. However, the pool of dGTP remains unchanged in the mutant-infected cells as opposed to a 200-fold increase in the wild-type phage-infected cells. Uninfected E. coli optA+ strains contain severalfold higher levels of dGTP compared to E. coli optA1 cells. In agreement with this observation, dGTP can fully substitute for purified gene 1.2 protein in restoring DNA packaging activity to extracts prepared from E. coli optA1 cells infected with T7 gene 1.2 mutants. dGMP or polymers containing deoxyguanosine can also restore packaging activity while dGDP is considerably less effective. dATP, dTTP, dCTP, and ribonucleotides have no significant effect. The addition of dGTP or dGMP to packaging extracts restores DNA synthesis. Gene 1.2 protein elevates the level of dGTP in these packaging extracts and restores DNA synthesis, thus suggesting that depletion of a guanine deoxynucleotide pool in E. coli optA1 cells infected with T7 gene 1.2 mutants may account for the observed defects.

DNA Replication↗

PubMed1987

Enzymatic properties of a proteolytically nicked RNA polymerase of bacteriophage T7.

The RNA polymerase of bacteriophage T7 is sensitive to cleavage by a protease associated with the membrane fraction of many strains of Escherichia coli. A major degradation product is a T7 RNA polymerase that is proteolytically cleaved between amino acids 172 (lysine) and 173 (arginine) (Tabor, S., and Richardson, C.C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074-1078). The cleavage splits the enzyme into a large fragment (Mr approximately 75,000) and a small fragment (Mr approximately 23,000) which remain tightly associated during the purification of nicked RNA polymerase. The protein retains RNA polymerase activity, but specific activity is reduced 3.5-fold. The proteolytic cleavage also reduces the Mg2+ requirements, increases the apparent Michaelis-Menten constants for the utilization of the ribonucleoside 5'-triphosphates, increases the temperature sensitivity, increases the sensitivity to inhibition by heparin, and increases the probability that a transcript will not be removed from the template. The reduced activity of nicked T7 RNA polymerase is apparently a consequence of inefficient initiation and premature termination. Nicked T7 RNA polymerase successfully initiates at the phi 10 promoter at half the efficiency of native T7 RNA polymerase. Transcripts synthesized by the nicked enzyme are also significantly shorter than transcripts synthesized by the native enzyme. In contrast, nicked T7 RNA polymerase and T7 RNA polymerase exhibit equivalent poly(dI).poly(dC)-dependent activity and equivalent polymerization velocities (60 bases/s at 25 degrees C).

Cell Membrane↗

PubMed1987

Interactions of a proteolytically nicked RNA polymerase of bacteriophage T7 with its promoter.

The association of nicked RNA polymerase of bacteriophage T7 (Ikeda, R. A., and Richardson, C. C. (1987) J. Biol. Chem. 262, 3790-3799) with the T7 phi 10 promoter has been examined by DNA cleavage protection. The phi 10 promoter consists of a 23-base pair consensus sequence that extends from -17 to +6 with respect to the site of the initiation of transcription (+1). Nicked T7 RNA polymerase alone protects 20 bases from -21 to -2 (+/- 1) base at each border. Initiation and synthesis of the trinucleotide r(GGG) expands and shifts the sequence protected by nicked T7 RNA polymerase. Twenty-five bases are protected from -17 to +8 (+/- 1). The polymerization of three additional ribonucleotides, synthesis of the hexamer r(GGGAGA), further expands the protected sequence. Twenty-seven bases are protected from -17-+10 (+/- 1). Finally, the synthesis of a pentadecaribonucleotide transcript, r(GGGAGACCACGG), leads to the formation of a transcription complex that protects 22 bases from -2-+20 (+/- 1). In comparison to the sequences protected by T7 RNA polymerase the sequences protected by the nicked enzyme are shortened at the 5' end and are translocated downstream much earlier during the initiation of transcription. It appears that a portion of the DNA contacts made at the amino terminus of T7 RNA polymerase are disrupted in the small fragment of nicked T7 RNA polymerase. The changes that are observed in the sequences protected by nicked T7 RNA polymerase are reflected in the physical characteristics of the DNA X enzyme complexes. The number of ion pairs formed by the r(GGG)-initiated complex of the nicked enzyme is reduced, and the association constant for the formation of the r(GGG)-initiated complex is decreased as compared to the intact T7 RNA polymerase.

Base Sequence↗

PubMed1987

DNA sequence analysis with a modified bacteriophage T7 DNA polymerase.

A chemically modified phage T7 DNA polymerase has three properties that make it ideal for DNA sequencing by the chain-termination method. The enzyme is highly processive, catalyzing the polymerization of thousands of nucleotides without dissociating. By virtue of the modification the 3' to 5' exonuclease activity is eliminated. The modified polymerase efficiently uses nucleotide analogs that increase the electrophoretic resolution of bands in gels. Consequently, dideoxynucleotide-terminated fragments have highly uniform radioactive intensity throughout the range of a few to thousands of nucleotides in length. There is virtually no background due to terminations at pause sites or secondary-structure impediments. Processive synthesis with dITP in place of dGTP eliminates band compressions, making possible the unambiguous determination of sequences from a single orientation.

Base Sequence↗

PubMed1986

Interaction of mutant thioredoxins of Escherichia coli with the gene 5 protein of phage T7. The redox capacity of thioredoxin is not required for stimulation of DNA polymerase activity.

DNA polymerase activity in Escherichia coli cells infected with bacteriophage T7 resides in a protein complex consisting of the T7 gene 5 protein and E. coli thioredoxin in a 1 to 1 stoichiometry. We have analyzed nine mutant thioredoxins, both in vivo and in vitro, for their ability to interact with the T7 gene 5 protein and stimulate the DNA polymerase and exonuclease activities inherent in gene 5 protein. The efficiency of plating of T7 on E. coli thioredoxin mutants depends strongly on the copy number of the respective mutant thioredoxin allele. Plating efficiencies at a constant copy number correlate well with the affinity of the purified mutant proteins for T7 gene 5 protein. The observed dissociation constant, Kobs, is increased between 5 and several hundredfold at 42 degrees C compared to wild-type thioredoxin. The maximum polymerase activity of the reconstituted gene 5 protein-thioredoxin complex at saturating concentrations of mutant thioredoxins, however, is reduced by less than 20%. Consequently, none of the mutant thioredoxins acts as a competitive inhibitor of wild-type thioredoxin. The active-site disulfide of thioredoxin is not essential for the activities of the gene 5 protein-thioredoxin complex. Both cysteines can be replaced without significantly affecting the maximum polymerase or exonuclease activities. Substitution or alkylation of either cysteine, however, reduces the affinity for gene 5 protein drastically, indicating that the active site is part of the thioredoxin surface involved in the protein-protein interaction.

Bacterial Proteins↗

PubMed1986

Interactions of the DNA polymerase and gene 4 protein of bacteriophage T7. Protein-protein and protein-DNA interactions involved in RNA-primed DNA synthesis.

Three proteins catalyze RNA-primed DNA synthesis on the lagging strand side of the replication fork of bacteriophage T7. Oligoribonucleotides are synthesized by T7 gene 4 protein, which also provides helicase activity. DNA synthesis is catalyzed by gene 5 protein of the phage, and processivity of DNA synthesis is conferred by Escherichia coli thioredoxin, a protein that is tightly associated with gene 5 protein. T7 DNA polymerase and gene 4 protein associate to form a complex that can be isolated by filtration through a molecular sieve. The complex is stable in 50 mM NaCl but is dissociated by 100 mM NaCl, a salt concentration that does not inhibit RNA-primed DNA synthesis. T7 DNA polymerase forms a stable complex with single-stranded M13 DNA at 50 mM NaCl as measured by gel filtration, and this complex requires 200 mM NaCl for dissociation, a salt concentration that inhibits RNA-primed DNA synthesis. Gene 4 protein alone does not bind to single-stranded DNA. In the presence of MgCl2 and dTTP or beta, gamma-methylene dTTP, a gene 4 protein-M13 DNA complex that is stable at 200 mM NaCl is formed. The affinity of DNA polymerase for both gene 4 protein and single-stranded DNA leads to the formation of a gene 4 protein-DNA polymerase-M13 DNA complex even in the absence of nucleoside triphosphates. However, the binding of each protein to DNA plays an important role in mediating the interaction of the proteins with each other. High concentrations of single-stranded DNA inhibit RNA-primed DNA synthesis by diluting the amount of proteins bound to each template and reducing the frequency of protein-protein interactions. Preincubation of gene 4 protein, DNA polymerase, and M13 DNA in the presence of dTTP forms protein-DNA complexes that most efficiently catalyze RNA-primed DNA synthesis in the presence of excess single-stranded competitor DNA.

DNA Replication↗

PubMed1986

Dissection of RNA-primed DNA synthesis catalyzed by gene 4 protein and DNA polymerase of bacteriophage T7. Coupling of RNA primer and DNA synthesis.

Gene 4 protein and DNA polymerase of bacteriophage T7 catalyze RNA-primed DNA synthesis on single-stranded DNA templates. T7 DNA polymerase exhibits an affinity for both gene 4 protein and single-stranded DNA, and gene 4 protein binds stably to single-stranded DNA in the presence of dTTP (Nakai, H. and Richardson, C. C. (1986) J. Biol. Chem. 261, 15208-15216). Gene 4 protein-T7 DNA polymerase-template complexes may be formed in both the presence and absence of nucleoside 5'-triphosphates. The protein-template complexes may be isolated free of unbound proteins and nucleotides by gel filtration and will catalyze RNA-primed DNA synthesis in the presence of ATP, CTP, and the four deoxynucleoside 5'-triphosphates. RNA-primed DNA synthesis may be dissected into separate reactions for primer synthesis and DNA synthesis. Upon incubation of gene 4 protein with single-stranded DNA, ATP, and CTP, a primer-template complex is formed; it is likely that gene 4 protein mediates stable binding of the oligonucleotide to the template. The complex, purified free of unbound proteins and nucleotides, supports DNA synthesis upon addition of DNA polymerase and deoxynucleoside 5'-triphosphates. Association of primers with the template is increased by the presence of dTTP or DNA polymerase during primer synthesis. DNA synthesis supported by primer-template complexes initiates predominantly at gene 4 recognition sequences, indicating that primers are bound to the template at these sites.

Base Sequence↗

PubMed1986

Interactions of the RNA polymerase of bacteriophage T7 with its promoter during binding and initiation of transcription.

Promoters for T7 RNA polymerase have a highly conserved sequence of 23 continuous base pairs located at position -17 to +6 relative to the initiation site for the RNA. The complex of T7 RNA polymerase with the phage phi 10 promoter has been visualized indirectly by exploiting the ability of the polymerase to protect DNA sequences from cleavage by methidiumpropyl-EDTA X Fe(II). The DNA contacts made by T7 RNA polymerase have been mapped during binding and during the subsequent initiation of transcription. The RNA polymerase alone protects 19 bases in a region from -21 to -3. Synthesis of the trinucleotide r(GGG) expands the length of the sequence protected by the RNA polymerase and stabilizes the complex; 29 bases (-21 to +8) are protected, and the observed equilibrium association constant of the r(GGG) complex is 5 X 10(5) M-1. The formation of a hexanucleotide mRNA, r(GGGAGA), further extends the protected region; 32 bases (-21 to +11) are protected. Finally, the synthesis of a pentadecanucleotide mRNA leads to a translocation of the region protected by the protein; the sequence now protected is reduced to 24 bases (-4 to +20).

Base Sequence↗

PubMed1985

Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins. Site and direction of initial DNA synthesis.

In vivo, T7 DNA replication is initiated 15% of the distance from the genetic left end of the chromosome. This site, the primary origin of replication, consists of a 200-base pair (bp) intergenic segment from 14.5 to 15.0% within which are located two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by a 61-bp AT-rich (79% A + T) region. A fragment of T7 DNA containing the primary origin has been inserted into plasmids in order to facilitate studies on initiation in vitro. Initiation of DNA synthesis can be reconstituted using T7 RNA polymerase, T7 DNA polymerase, and T7 origin-containing plasmid DNAs. DNA synthesis is stimulated greatly by the T7 gene 4 protein, an enzyme that has helicase and primase activities. When T7 gene 4 protein is present, replication primarily yields partially replicated Y-form molecules as observed by electron microscopy. Synthesis is unidirectional and the branches of the Y-form molecules are uniform in size, with the branch point of the Y located at the origin. Using restriction enzyme analysis, DNA synthesis has been shown to proceed in the same direction (rightward with respect to the T7 genetic map) as transcription from the two promoters located at the origin. Initiation of DNA synthesis in the opposite direction requires the addition of a single-stranded DNA-binding protein (Fuller, C.W., and Richardson, C.C. (1985) J. Biol. Chem. 260, 3197-3206). The initial products of DNA synthesis have been analyzed by polyacrylamide gel electrophoresis. These DNAs have 10 to 60 ribonucleotides covalently linked to their 5' termini. These RNA primers arise by transcription from each of the two promoters, phi 1.1A and phi 1.1B, located within the primary origin.

Base Sequence↗

PubMed1985

Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins. Initiation of bidirectional synthesis.

Replication of bacteriophage T7 DNA initiates in vivo at an origin located 15% of the distance from the genetic left end of the chromosome. Bidirectional DNA synthesis from this site results in complete replication of the chromosome. The combination of T7 RNA polymerase, T7 DNA polymerase, and T7 gene 4 protein initiates DNA synthesis in vitro within the cloned origin sequence (Fuller, C. W., and Richardson, C. C. (1985) J. Biol. Chem. 260: 3185-3196). DNA synthesis is primed by T7 RNA polymerase transcripts, and proceeds in the same direction (rightward) as transcription to yield partially replicated Y-form DNA molecules. The DNA product of in vitro synthesis (Y-form DNA) has been characterized by electron microscopic, sedimentation, and gel electrophoretic analyses. These studies show that Y-form DNA is the product of unidirectional replication of both leading and lagging strands from the origin to the right-hand end of the template. The inclusion of either Escherichia coli single-stranded DNA-binding protein or the functionally similar T7 gene 2.5 protein results in marked stimulation of bidirectional synthesis. Studies using purified Y-form DNA provide direct evidence that this species is an intermediate in the complete replication of the linear template. Purified Y-form DNA is converted to linear DNA in a reaction catalyzed by T7 DNA polymerase, T7 gene 4 protein, and single-stranded DNA-binding protein. Y-form DNA is a competent, transient intermediate during the bidirectional replication of linear DNA molecules and DNA-binding protein is essential to initiate leftward synthesis.

Centrifugation, Density Gradient↗

PubMed1985

Nucleotide-dependent binding of the gene 4 protein of bacteriophage T7 to single-stranded DNA.

The gene 4 protein of bacteriophage T7 is a multifunctional enzyme that catalyzes (i) the hydrolysis of nucleoside 5'-triphosphates, (ii) the synthesis of tetraribonucleotide primers at specific recognition sequences on a DNA template, and (iii) the unwinding of duplex DNA. All three activities depend on binding of gene 4 protein to single-stranded DNA followed by unidirectional 5' to 3' translocation of the protein (Tabor, S., and Richardson, C. C. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 205-209). Binding of gene 4 protein to single-stranded DNA, assayed by retention of DNA-protein complexes on nitrocellulose filters, is random with regard to DNA sequence. Although gene 4 protein does not bind to duplex DNAs, the presence of a 240-nucleotide-long single-stranded tail on a 7200-base pair duplex DNA molecule is sufficient for gene 4 protein to cause retention of the DNA on a filter. The binding reaction requires, in addition to MgCl2, the presence of a nucleoside 5'-triphosphate, but binding is not dependent on hydrolysis; nucleoside 5'-diphosphate will substitute for nucleoside 5'-triphosphate. Of the eight common nucleoside triphosphates, dTTP promotes optimal binding. The half-life of the gene 4 protein-DNA complex depends on both the secondary structure of the DNA and on whether or not the nucleoside 5'-triphosphate cofactor can be hydrolyzed. Using the nonhydrolyzable nucleoside 5'-triphosphate analog, beta,gamma-methylene dTTP, the half-life of the gene 4 protein-DNA complex is greater than 80 min. In the presence of the hydrolyzable nucleoside 5'-triphosphate, dTTP, the half-life of the gene 4 protein-DNA complex using circular M13 DNA is at least 4 times longer than that observed using linear M13 DNA.

Base Sequence↗

PubMed1985

A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes.

The RNA polymerase gene of bacteriophage T7 has been cloned into the plasmid pBR322 under the inducible control of the lambda PL promoter. After induction, T7 RNA polymerase constitutes 20% of the soluble protein of Escherichia coli, a 200-fold increase over levels found in T7-infected cells. The overproduced enzyme has been purified to homogeneity. During extraction the enzyme is sensitive to a specific proteolysis, a reaction that can be prevented by a modification of lysis conditions. The specificity of T7 RNA polymerase for its own promoters, combined with the ability to inhibit selectively the host RNA polymerase with rifampicin, permits the exclusive expression of genes under the control of a T7 RNA polymerase promoter. We describe such a coupled system and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase.

Bacteriophage lambda↗