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L B Rothman-Denes

Publications and source records attributed to L B Rothman-Denes.

At least 37 records · Page 2Linked to original sources
PubMed1989

In vitro requirements for N4 RNA polymerase II-specific initiation.

The synthesis of coliphage N4 middle RNAs requires the activity of three phage-coded proteins, P7, P4, and P17, of molecular weights 40,000, 30,000, and 15,000, respectively. P4 and P7 constitute a heterodimeric RNA polymerase, N4 RNA polymerase II, which cannot transcribe or bind to native N4 DNA. Denatured templates are transcribed but without specificity. We present the development of an in vitro transcription system, composed of purified N4 RNA polymerase II and solubilized P17, which accurately utilizes in vivo sites of transcription initiation. Possible mechanisms for P17 action are discussed.

Base Sequence↗
PubMed1989

Purification and characterization of the coliphage N4-coded single-stranded DNA binding protein.

We have purified and characterized a single-stranded DNA binding protein (N4 SSB) induced after coliphage N4 infection. It has a monomeric molecular weight of 31,000 and contains 10 tyrosine and 1-2 tryptophan amino acid residues. Its fluorescence spectrum is dominated by the tyrosine residues, and their fluorescence is quenched when the protein binds single-stranded DNA. Fluorescence quenching was used as an assay to quantitate binding of the protein to single-stranded nucleotides. The N4 single-stranded DNA binding protein binds cooperatively to single-stranded nucleic acids and binds single-stranded DNA more tightly than RNA. The binding involves displacement of cations from the DNA and anions from the protein. The apparent binding affinity is very salt-dependent, decreasing as much as 1,000-fold for a 10-fold increase in NaCl concentration. The degree of cooperativity (omega) is relatively independent of salt concentration. At 37 degrees C in 0.22 M NaCl, the protein has an intrinsic binding constant for M13 viral DNA of 3.8 x 10(4) M-1, a cooperativity factor omega of 300, and binding site size of 11 nucleotides per monomer. The protein lowers the melting point of poly(dA.dT).poly(dA-dT) by greater than 60 degrees C but cannot lower the melting transition or assist in the renaturation of natural DNA. N4 single-stranded DNA binding protein enhances the rate of DNA synthesis catalyzed by the N4 DNA polymerase by increasing the processivity of the N4 DNA polymerase and melting out hairpin structures that block polymerization.

Chromatography, Affinity↗
PubMed1989

Genetic analysis of bacteriophage N4 adsorption.

We isolated six mutants of Escherichia coli K-12 that were defective in bacteriophage N4 adsorption. We mapped the mutations to four loci designated nfrA through nfrD (N four resistance). nfrA and nfrB were tightly linked to each other and were mapped to min 12 of the E. coli linkage map. nfrC was mapped to min 85, and nfrD was mapped between min 44 and 58. We isolated a clone carrying both nfrA and nfrB and identified its gene products through maxicell analysis of plasmid subclones. The nfrA gene product was an outer membrane protein of 96,000 apparent molecular weight, whereas nfrB encoded an inner-membrane protein of 69,500 apparent molecular weight. The nfrB1 mutation did not affect the export of the nfrA gene product to the outer membrane and did not affect the alkaline phosphatase activity of an nfrA-phoA fusion. We propose that nfrA encodes the structural receptor for N4 and that the nfrB gene product may be required for irreversible adsorption and injection of the phage genome and virion-encapsulated RNA polymerase through the inner membrane.

Alleles↗
PubMed1988

Purification and characterization of bacteriophage N4-induced DNA polymerase.

Coliphage N4 replication is independent of most host DNA replication functions except for the 5'----3' exonuclease activity of polA, DNA ligase, DNA gyrase, and ribonucleotide reductase (Guinta, D., Stambouly, J., Falco, S. C., Rist, J. K., and Rothman-Denes, L. B. (1986) Virology 150, 33-44). It is therefore expected that N4 codes for most of the functions required for replication of its genome. In this paper we report the purification of the N4-coded DNA polymerase from N4-infected cell extracts by following its activity on a gapped template and in an in vitro complementation system for N4 DNA replication (Rist, J. K., Pearle, M., Sugino, A., and Rothman-Denes, L. B. (1986) J. Biol. Chem. 261, 10506-10510). The enzyme is composed of one polypeptide, Mr 87,000. It is most active on templates containing short gaps synthesizing DNA with high fidelity in a quasi-processive manner. A strong 3'----5' exonuclease activity is associated with the DNA polymerase polypeptide. No 5'----3' exonuclease or strand-displacing activities were detected.

Coliphages↗
PubMed1988

Structure of the ends of the coliphage N4 genome.

The coliphage N4 genome, a linear and double-stranded DNA of approximately 72,000 bases in length, has unique (non-permuted) direct terminal repeats of 390 to 440 base-pairs in length with 3' extensions. The very terminal sequences were determined by the Maxam-Gilbert method after 5' or 3' labeling, while sequences of internal fragments were determined by the dideoxy chain terminator method after cloning them onto M13 phage DNA. The left end of the N4 genome is relatively precise at its 5' terminus, while microheterogeneity of length exists at the 3'-terminal extensions. The predominant species had a 5 or 6 base 3' protruding sequence, 3' CATAA or 3' CATAAA. On the other hand, the right end is variable; there are at least six discrete ends differing from each other by approximately ten base-pairs and giving rise to the variability of the length of the terminal repeats. Each of the six discrete ends has a microheterogeneity of length, especially at the 3' termini. These properties of the terminal redundancy are discussed in conjunction with the mechanism whereby N4 DNA is replicated and processed.

Base Sequence↗
PubMed1988

N4 virion DNA dependent-RNA polymerase: initiation sequences utilized by the enzyme on heterologous templates.

Bacteriophage N4 virion-encapsulated RNA polymerase, the enzyme responsible for transcription of the phage early RNAs, is unable to use duplex linear DNA as a template. In contrast to other RNA polymerases, the enzyme transcribes denatured N4 DNA with in vivo specificity. The promoter sequences for three sites of transcription initiation on the N4 genome have been determined and found to contain conserved sequences and two sets of inverted repeats. In order to define the minimal sequence requirements for N4 virion RNA polymerase activity, we have screened several heterologous DNAs, amounting to 64,328 bases, for their ability to support transcription. Several sequences allowing specific initiation were found. Their location, properties and the relation to N4 virion RNA polymerase promoters are discussed.

Coliphages↗
PubMed1988

Cloning and generation of a genetic map of bacteriophage N4 DNA.

Analysis of coliphage N4 development has been hindered by the lack of a genetic map. Conventional methods of complementation and recombination have been inadequate because N4 shows very high levels of recombination, which are independent of the host recombination system. We have cloned restriction fragments of the 72-kb genome of N4 and have used these clones to rescue a collection of suppressor-sensitive and temperature-sensitive mutants. After mutations were localized to a small region by marker rescue, complementation groups were defined. In this way several functions essential for transcription and replication have been mapped. Finally, a nonessential 6-kb region of the N4 genome has been identified by characterizing phage deletions isolated after heat and citrate treatment of virions.

Chromosome Deletion↗
PubMed1986

Development of an in vitro bacteriophage N4 DNA replication system.

An in vitro DNA replication system from bacteriophage N4-infected Escherichia coli has been developed. It requires MgCl2, all four deoxyribonucleoside triphosphates, and exogenously added N4 phage DNA; other DNAs are used inefficiently or not at all. Ribonucleoside triphosphates are not required, although they stimulate DNA synthesis. In vitro replication starts at the ends of the N4 genome and moves progressively inward. Initiation occurs through hairpin priming at the 3' ends of the genome, but shows a strong preference for the right end. Three N4 gene products (dnp, dbp, and exo) required in vivo for N4 DNA synthesis are absolutely required in the in vitro system. These findings are discussed with respect to the mode of N4 DNA replication.

Coliphages↗
PubMed1986

Bacteriophage N4-coded 5'----3' exonuclease. Purification and characterization.

Bacteriophage N4 DNA replication requires the activity of a phage-induced exonuclease. We show here that the activity is phage coded. We have purified this enzyme to apparent homogeneity. It has a denatured molecular weight of 45,000 and exists in solution as a dimer. Duplex DNA is the preferred substrate which it degrades in a 5'----3' direction to 5' mononucleotides by a distributive mechanism. The enzyme does not act at a nick or a gap; indeed, it requires an end for activity. A possible role for this exonuclease in N4 replication is discussed.

Coliphages↗
PubMed1986

Host and phage-coded functions required for coliphage N4 DNA replication.

Escherichia coli strains containing mutations in various deoxyribonucleic acid synthesis cistrons have been tested for their ability to support bacteriophage N4 growth and, specifically, N4 DNA synthesis. N4 DNA synthesis is independent of the activity of the products of the E. coli dnaA, dnaB, dnaC, dnaE, dnaG, and rep genes. In contrast, N4 DNA replication requires the products of the dnaF, (ribonucleotide reductase) and lig (DNA ligase) genes of E. coli. N4 DNA replication, specifically processing of short DNA fragments requires the 5'-3' exonuclease activity of the polA gene product. However, its DNA polymerizing activity is not required. In addition, the sensitivity of N4 DNA synthesis to inhibitors or temperature-sensitive mutants of E. coli DNA gyrase suggests that this activity is required for N4 DNA synthesis. To date, we have found five N4 gene products required for N4 DNA replication: dbp (a single-stranded DNA binding protein), dnp (a DNA polymerase), dns (unknown function), vRNAp (the N4 virion-associated, DNA-dependent RNA polymerase) and exo (a 5'-3' exonuclease).

Coliphages↗
PubMed1986

Cell type-specific negative regulatory element in the control region of the rat alpha-fetoprotein gene.

Albumin and alpha-fetoprotein are evolutionarily related genes that show differential and complex patterns of regulation during development. We investigated the role of the sequences flanking the transcription initiation site of the rat alpha-fetoprotein gene in transient transfection assays of hepatic and nonhepatic cell lines. Chimeric flanking regions and deletion analysis have defined the following three functionally different regions: a cell type-specific enhancer(s), encompassing 3 kilobases, located between -7 kilobase pairs and -4 kilobase pairs; a cell type-specific promoter, inactive in the absence of an enhancer and comprising at most the 180 base pairs upstream from the site of transcription initiation; and a 550-base-pair region, located between the promoter and the enhancer (at -3.5 kilobases), down-regulates transcription initiation in a cell type-specific manner and is also capable of repressing heterologous promoters. The implications of these findings with respect to the complex pattern of AFP regulation are discussed.

Albumins↗
PubMed1985

N4 virion RNA polymerase sites of transcription initiation.

Coliphage N4 virion encapsulated RNA polymerase shows a marked preference for denatured N4 DNA as a template. We show that initiation on denatured N4 virion DNA occurs with in vivo specificity. The location of the in vivo and in vitro initiation sites and the corresponding DNA sequences were determined. The N4 virion RNA polymerase promoters contain extensive sequence homology from position -18 to position 1, with a conserved GC-rich heptamer centered at -12, and two sets of short inverted repeats. We suggest that the N4 virion RNA polymerase recognizes the promoter only in a novel single-stranded form, and that the formation of the initiation complex is facilitated in vivo by supercoiling and E. coli single-stranded DNA binding protein.

Base Sequence↗
PubMed1983

Purification and characterization of coliphage N4 RNA polymerase II activity from infected cell extracts.

The soluble components of the RNA polymerase activity (N4 RNA polymerase II) required for coliphage N4 middle RNA synthesis have been purified to homogeneity using a complementation assay described elsewhere. These soluble components are found to exhibit the properties of a DNA-dependent RNA polymerase which is resistant to both rifampicin and streptolydigin and transcribes denatured N4 DNA with marked preference but with little selectivity for the middle region of the N4 genome. In its native form, the activity is composed of one Mr = 40,000 polypeptide (p4, the product of N4 cistron 4) and one Mr = 30,000 polypeptide (p7, the product of N4 cistron 3). Its physical properties and the mechanism of transcription selectivity are discussed.

Coliphages↗
PubMed1983

Bacteriophage N4-induced transcribing activities in E. coli. III. A third cistron required for N4 RNA polymerase II activity.

The requirement of a third gene product of molecular weight 15,000 for coliphage N4 middle RNA synthesis is described. Therefore, three proteins of molecular weights 15,000, 30,000, and 40,000, which constitute the N4 RNA polymerase II activity, are required for N4 middle RNA synthesis. A complementation assay for N4 RNA polymerase II activity, which requires a DNA-membrane complex carrying the 15,000-MW protein and a supernatant providing the 30,000- and 40,000-MW proteins, has been developed. This assay, which reproduces the requirement for the cistron 5 gene product in vitro provides a means for the purification of the soluble components of the N4 RNA polymerase II activity (to be reported elsewhere). The cistron 5 gene product is also required for N4 RNA polymerase II to synthesize RNAs utilizing middle promoters resident in recombinant plasmids. Mechanisms by which the cistron 5 gene product affects N4 RNA polymerase II transcription in vivo and in vitro are discussed.

Coliphages↗
PubMed1980

DNA-dependent RNA polymerase from bacteriophage N4 virions. Purification and characterization.

A DNA-dependent RNA polymerase has been purified to homogeneity from bacteriophage N4 virions. Sodium dodecyl sulfate-8 M urea polyacrylamide gel electrophoresis of the enzyme revealed a single polypeptide with a molecular weight of 350,000. The hydrodynamic properties of the enzyme have been determined to be 9.5 S for the sedimentation coefficient and 84 A for the Stokes radius. These two parameters indicate a native molecular weight of 320,000. Enzyme activity is dependent on the presence of Mg2+, the four ribonucleoside triphosphates, and denatured N4 DNA. Under these conditions, initiation of RNA synthesis occurs exclusively with pppG. The enzyme is inhibited by monovalent salts and is resistant to the drugs rifampicin and streptolydigin. The protein is present in 1 to 2 copies per virion; its presence provides an explanation for the independence of N4 early RNA synthesis on the activity of the host RNA polymerase.

Antimetabolites↗