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L B Rothman-Denes

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

At least 19 recordsLinked to original sources
PubMed2002

The phage N4 virion RNA polymerase catalytic domain is related to single-subunit RNA polymerases.

In vitro, bacteriophage N4 virion RNA polymerase (vRNAP) recognizes in vivo sites of transcription initiation on single-stranded templates. N4 vRNAP promoters are comprised of a hairpin structure and conserved sequences. Here, we show that vRNAP consists of a single 3500 amino acid polypeptide, and we define and characterize a transcriptionally active 1106 amino acid domain (mini-vRNAP). Biochemical and genetic characterization of this domain indicates that, despite its peculiar promoter specificity and lack of extensive sequence similarity to other DNA-dependent RNA polymerases, mini-vRNAP is related to the family of T7-like RNA polymerases.

Amino Acid Motifs↗
PubMed2002

N4 RNA polymerase II, a heterodimeric RNA polymerase with homology to the single-subunit family of RNA polymerases.

Bacteriophage N4 middle genes are transcribed by a phage-coded, heterodimeric, rifampin-resistant RNA polymerase, N4 RNA polymerase II (N4 RNAPII). Sequencing and transcriptional analysis revealed that the genes encoding the two subunits comprising N4 RNAPII are translated from a common transcript initiating at the N4 early promoter Pe3. These genes code for proteins of 269 and 404 amino acid residues with sequence similarity to the single-subunit, phage-like RNA polymerases. The genes encoding the N4 RNAPII subunits, as well as a synthetic construct encoding a fusion polypeptide, have been cloned and expressed. Both the individually expressed subunits and the fusion polypeptide reconstitute functional enzymes in vivo and in vitro.

Amino Acid Sequence↗
PubMed2001

A genomic approach to gene fusion technology.

Gene expression profiling provides powerful analyses of transcriptional responses to cellular perturbation. In contrast to DNA array-based methods, reporter gene technology has been underused for this application. Here we describe a genomewide, genome-registered collection of Escherichia coli bioluminescent reporter gene fusions. DNA sequences from plasmid-borne, random fusions of E. coli chromosomal DNA to a Photorhabdus luminescens luxCDABE reporter allowed precise mapping of each fusion. The utility of this collection covering about 30% of the transcriptional units was tested by analyzing individual fusions representative of heat shock, SOS, OxyR, SoxRS, and cya/crp stress-responsive regulons. Each fusion strain responded as anticipated to environmental conditions known to activate the corresponding regulatory circuit. Thus, the collection mirrors E. coli's transcriptional wiring diagram. This genomewide collection of gene fusions provides an independent test of results from other gene expression analyses. Accordingly, a DNA microarray-based analysis of mitomycin C-treated E. coli indicated elevated expression of expected and unanticipated genes. Selected luxCDABE fusions corresponding to these up-regulated genes were used to confirm or contradict the DNA microarray results. The power of partnering gene fusion and DNA microarray technology to discover promoters and define operons was demonstrated when data from both suggested that a cluster of 20 genes encoding production of type I extracellular polysaccharide in E. coli form a single operon.

Artificial Gene Fusion↗
PubMed1999

DNA structure and transcription.

Regulation of transcription occurs through complex interactions of RNA polymerase and accessory proteins with specific DNA sequences and with each other. The DNA template topology influences the interaction of RNA polymerase with the promoter and its response to repressors, activators and the intracellular milieu through the formation of altered DNA structures or of nucleoprotein complexes. Recent developments on the role of DNA structures in transcription regulation are discussed.

DNA↗
PubMed1998

Sequence and DNA structural determinants of N4 virion RNA polymerase-promoter recognition.

Coliphage N4-coded, virion-encapsidated RNA polymerase (vRNAP) is able to bind to and transcribe promoter-containing double-stranded DNAs when the template is supercoiled and Escherichia coli single-stranded DNA-binding protein (Eco SSB) is present. We report that vRNAP-promoter recognition and activity on these templates require specific sequences and a hairpin structure on the template strand. Hairpin extrusion, induced by Mg(II) and physiological superhelical density, is essential to provide the correct DNA structure for polymerase recognition, as mutant promoters that do not form hairpins show reduced in vitro activity. Therefore, a supercoil-induced DNA structural transition regulates N4 vRNAP transcription. Eco SSB activates transcription at physiological superhelical densities by stabilizing the template-strand hairpin. Specific sequences at the promoters are conserved to provide proper contacts for vRNAP, to support hairpin extrusion, or both. We propose a model for in vivo utilization of the vRNAP promoters, and discuss the roles of DNA supercoiling and Eco SSB in promoter activation.

Base Sequence↗
PubMed1998

Sequence-dependent extrusion of a small DNA hairpin at the N4 virion RNA polymerase promoters.

Bacteriophage N4 virion RNA polymerase promoters contain five to seven-base inverted repeats separated by three bases and centered at position -12 from the site of transcription initiation. We have previously shown that these inverted repeats extrude as hairpins at physiological superhelical densities in a Mg(II)-dependent manner. Mg(II)-dependent hairpin extrusion at promoters P1 and P2 displays quantitative differences in reactivity to structural probes at different DNA superhelical densities, with extrusion at P2 being more favored at low superhelical density. Analyses of mutant promoters using structure-specific probes revealed that specific sequences, at the closing base-pair of the hairpin and at the loop (i.e. 5'-C-GXA-G-3' where X=G, A, T), are required for extrusion of the small promoter hairpins at physiological superhelical density. The sequence-dependent requirements for extrusion of the small N4 promoter hairpins may be generally applicable for other such sequences found both in prokaryotic and eukaryotic genomes.

Acetaldehyde↗
PubMed1997

Supercoil-induced extrusion of a regulatory DNA hairpin.

Bacteriophage N4 virion RNA polymerase (N4 vRNAP) promoters contain inverted repeats, which form a 5- to 7-base-pair stem, 3-base loop hairpin that is required for vRNAP recognition. We show that, contrary to certain theoretical predictions, hairpin extrusion can occur at physiological superhelical densities in a Mg2+-dependent manner. Specific sequences on the template strand are required for hairpin extrusion. These sequences define stable DNA hairpins that are relatively unreactive to single strand-specific probes. In addition, a specific stable hairpin-inducing sequence can regulate transcription in vivo. Thus, a DNA structure, in its natural environment, is involved in transcriptional regulation.

Base Composition↗
PubMed1997

RNA polymerase beta' subunit: a target of DNA binding-independent activation.

The bacteriophage N4 single-stranded DNA binding protein (N4SSB) activates transcription by the Escherichia coli final sigma70-RNA polymerase at N4 late promoters. Here it is shown that the single-stranded DNA binding activity of N4SSB is not required for transcriptional activation. N4SSB interacts with the carboxyl terminus of the RNA polymerase beta' subunit in a region that is highly conserved in the largest subunits of prokaryotic and eukaryotic RNA polymerases.

Amino Acid Sequence↗
PubMed1996

E. coli SSB activates N4 virion RNA polymerase promoters by stabilizing a DNA hairpin required for promoter recognition.

Bacteriophage N4 virion RNA polymerase transcription of double-stranded promoter-containing DNAs requires supercoiled template and E. coli single-stranded DNA-binding protein (EcoSSB); other single-stranded DNA-binding proteins cannot substitute. The DNA determinants of virion RNA polymerase binding at the promoter comprise a small template-strand hairpin. The requirement for EcoSSB is surprising, since single-stranded DNA-binding proteins destabilize hairpin structures. DNA footprinting of EcoSSB on wild-type and mutant promoters indicates that EcoSSB stabilizes the template-strand hairpin owing to the hairpin-loop sequences. Other single-stranded DNA-binding proteins destabilize the promoter hairpin, explaining the specificity of EcoSSB activation. We conclude that EcoSSB activates transcription by providing the appropriate DNA structure for polymerase binding. The existence of small hairpins stable to single-stranded protein binding suggests a novel mechanism that provides structural determinants for specific recognition in single-stranded DNA transactions by an otherwise nonspecific DNA-binding protein.

Base Sequence↗
PubMed1995

Identification, cloning, and characterization of the bacteriophage N4 gene encoding the single-stranded DNA-binding protein. A protein required for phage replication, recombination, and late transcription.

The coliphage N4-coded single-stranded DNA-binding protein (N4SSB) is essential for phage replication and for expression of the phage late genes, which are transcribed by the Escherichia coli sigma 70 RNA polymerase. As a first step in investigating the role of N4SSB in replication and transcriptional activation, we have identified and sequenced the N4SSB gene. The gene encodes a 265-amino acid protein with no apparent sequence homology to other single-stranded DNA-binding proteins. We present data indicating that N4SSB is also essential for phage recombination. Mutational analysis of the carboxyl terminus of the protein indicates that this region is required for protein-protein interactions with the N4 replication, N4 recombination, and E. coli transcriptional machineries, while the rest of the protein contains the determinants for single-stranded DNA binding.

Amino Acid Sequence↗
PubMed1995

The bacteriophage N4-coded single-stranded DNA-binding protein (N4SSB) is the transcriptional activator of Escherichia coli RNA polymerase at N4 late promoters.

Transcription of the 72kb linear double-stranded DNA genome of coliphage N4 is carried out by the sequential activity of three different RNA polymerases. Early and middle viral transcripts are synthesized by two phage-coded RNA polymerases while late transcription is carried out by the Escherichia coli sigma 70-RNA polymerase. We have determined the sequences and sites of initiation of several N4 late transcripts; N4 late promoters share weak homology with the E. coli sigma 70 promoter consensus sequence. Indeed, N4 late promoters are weak templates for the host enzyme. We present evidence that the phage-coded, single-stranded DNA-binding protein (N4SSB), a protein that is required for phage DNA replication and recombination and does not bind with sequence specificity to DNA, is the activator of E. coli RNA polymerase at late N4 promoters. Models for the mechanism of action of N4SSB as a transcriptional activator are discussed.

Base Sequence↗
PubMed1993

A cytoplasmic protein, NfrC, is required for bacteriophage N4 adsorption.

At least four genes are required for irreversible adsorption of bacteriophage N4. nfrA and nfrB have been characterized previously and encode an outer membrane protein and inner membrane protein, respectively. The nfrC gene product is characterized in detail in this study. We have mapped the nfrD locus to min 52 on the Escherichia coli linkage map. Maxicell analysis of nfrC and a null allele (nfrC2) cloned into a high-copy-number plasmid shows its gene product to be 42 kDa in size. We determined the nfrC nucleotide sequence which predicts a gene product of 42 kDa. Western blots (immunoblots) of Escherichia coli proteins after cellular fractionation show NfrC to be a cytoplasmic protein which is required for irreversible bacteriophage N4 adsorption, an event occurring at the cell surface.

Adsorption↗
PubMed1992

Specific sequences and a hairpin structure in the template strand are required for N4 virion RNA polymerase promoter recognition.

Coliphage N4 virion-encapsidated, DNA-dependent RNA polymerase (vRNAP) is inactive on double-stranded N4 DNA; however, denatured promoter-containing templates are accurately transcribed. We report that all determinants of vRNAP promoter recognition exist in the template strand, indicating that this enzyme is a site-specific, single-stranded DNA-binding protein. We show that conserved sequences and the integrity of inverted repeats present at the promoters are essential for activity, suggesting the necessity for specific secondary structure. Evidence for such a structure is presented. We propose a model for in vivo utilization of vRNAP promoters in which template negative supercoiling yields single-strandedness at the promoter to reveal the determinants of vRNAP binding. This structure is stabilized by the binding of E. coli single-stranded DNA-binding protein to yield an "activated promoter."

Affinity Labels↗
PubMed1992

Escherichia coli single-stranded DNA-binding protein is a supercoiled template-dependent transcriptional activator of N4 virion RNA polymerase.

Coliphage N4 is a double-stranded DNA virus that requires the sequential activity of three different RNA polymerases during infection. The N4 virion RNA polymerase, which is carried in the virion and is injected with the DNA at the start of infection, is responsible for the synthesis of N4 early RNAs. In vitro, the virion RNA polymerase can transcribe double-stranded N4 DNA accurately and efficiently but only when the DNA is denatured. We have shown previously that the activity of DNA gyrase is required for in vivo early N4 transcription. We report here that Escherichia coli single-stranded DNA-binding protein (SSB) is also required for N4 early transcription. In vitro, linear or relaxed templates cannot be activated by SSB; however, supercoiled template and SSB allow the virion polymerase to recognize its promoters on duplex DNA and activate transcription. The effects of supercoiling are limited to transcript initiation and are not required for transcript elongation. The activation is specific for SSB; no other single-stranded DNA-binding proteins can substitute. Therefore, SSB is one of a small number of proteins that function to stimulate both replication and transcription. The basis for the specificity of SSB, the mechanism of transcriptional activation by SSB and template supercoiling, and their role in the N4 transcriptional program during development are discussed.

Base Sequence↗
PubMed1990

N4 RNA polymerase II sites of transcription initiation.

The RNA polymerase responsible for the synthesis of coliphage N4 middle RNAs, N4 RNA polymerase II, is composed of two subunits of 30,000 and 40,000 molecular weight. It is the smallest DNA-dependent RNA polymerase characterized to date. We have determined the sequences surrounding the sites of in vivo transcription initiation for this enzyme. Two regions of sequence homology are present: a box at +1, 3' AAAT 5', and a box, 3' TTCTGGAC 5' at a variable distance (16 to 24 base-pairs) upstream from +1. Possible mechanisms for recognition of these sequences are discussed.

Base Sequence↗