Lucia B. Rothman-Denes

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.

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. colis 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.

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.

Insight into DNA and Protein Transport in Double-stranded DNA Viruses: The Structure of Bacteriophage N4

Bacteriophage N4 encapsidates a 3500-aa-long DNA-dependent RNA polymerase (vRNAP), which is injected into the host along with the N4 genome upon infection. The three-dimensional structures of wild-type and mutant N4 viruses lacking gp17, gp50, or gp65 were determined by cryoelectron microscopy. The virion has an icosahedral capsid with T=9 quasi-symmetry that encapsidates well-organized double-stranded DNA and vRNAP. The tail, attached at a unique pentameric vertex of the head, consists of a neck, 12 appendages, and six ribbons that constitute a non-contractile sheath around a central tail tube. Comparison of wild-type and mutant virus structures in conjunction with bioinformatics established the identity and virion locations of the major capsid protein (gp56), a decorating protein (gp17), the vRNAP (gp50), the tail sheath (gp65), the appendages (gp66), and the portal protein (gp59). The N4 virion organization provides insight into its assembly and suggests a mechanism for genome and vRNAP transport strategies utilized by this unique system.

Structural Basis for DNA-Hairpin Promoter Recognition by the Bacteriophage N4 Virion RNA Polymerase

Coliphage N4 virion-encapsidated RNA polymerase (vRNAP) is a member of the phage T7-like single-subunit RNA polymerase (RNAP) family. Its central domain (mini-vRNAP) contains all RNAP functions of the full-length vRNAP, which recognizes a 5 to 7 base pair stem and 3 nucleotide loop hairpin DNA promoter. Here, we report the X-ray crystal structures of mini-vRNAP bound to promoters. Mini-vRNAP uses four structural motifs to recognize DNA sequences at the hairpin loop and stem and to unwind DNA. Despite their low sequence similarity, three out of four motifs are shared with T7 RNAP that recognizes a double-stranded DNA promoter. The binary complex structure and results of engineered disulfide linkage experiments reveal that the plug and motif B loop, which block the access of template DNA to the active site in the apo-form mini-vRNAP, undergo a large-scale conformational change upon promoter binding, explaining the restricted promoter specificity that is critical for N4 phage early transcription.

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.

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.

Transcriptional map of bacteriophage N4. Location and polarity of N4 RNAs.

Abstract The strands of the four bacteriophage N4 DNA fragments derived by HhaI endonuclease digestion have been separated on agarose gels, and the eight fragments obtained have been related to the two complementary strands of N4 DNA. We have used the HpaI restriction endonuclease fragments (Zivin et al., 1980) and single-stranded fragments of HhaI-digested N4 DNA as probes to determine the temporal appearance, localization and polarity of N4 transcripts. These experiments have allowed us to localize on the N4 genome the two previously characterized classes (early and middle RNAs) of N4 rifampicin-resistant RNAs (Vander Laan et al., 1977) and to detect a new class of N4 RNAs, which are synthesized late in infection. The synthesis of late N4 RNAs is rifampicin sensitive and, as we show in this paper, requires the activity of the Escherichia coli RNA polymerase.

Novel transcribing activities in N4-infected Escherichia coli

Abstract N4 is a small virus containing double-stranded DNA of molecular weight 40 × 10 6 , active on Escherichia coli K12 strains. Analysis of N4 transcription in infected cells by pulse labeling and hybridization has revealed the presence of two new RNA polymcrizing activities. The first N4 activity is unprecedented. It appears in cells treated prior to infection with both rifampicin and chloramphenicol and, therefore, requires neither transcription nor translation of the phage chromosome. This transcribing activity may be due to a previously undescribed host enzyme. The second activity, maximal at 6 min after infection at 37°, is also rifampicin resistant, but its appearance requires the expression and function of at least two N4 genes.

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.